Sodium channel blockers

ABSTRACT

The present invention relates to sodium channel blockers. The present invention also relates to a variety of methods of treatment using the sodium channel blockers.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to sodium channel blockers. Thepresent invention also includes a variety of methods of treatment usingthese inventive sodium channel blockers.

[0003] 2. Description of the Background

[0004] The mucosal surfaces at the interface between the environment andthe body have evolved a number of “innate defense”, i.e., protectivemechanisms. A principal form of such innate defense is to cleanse thesesurfaces with liquid. Typically, the quantity of the liquid layer on amucosal surface reflects the balance between epithelial liquidsecretion, often reflecting anion (Cl⁻ and/or HCO₃ ⁻) secretion coupledwith water (and a cation counter-ion), and epithelial liquid absorption,often reflecting Na⁺ absorption, coupled with water and counter anion(Cl⁻ and/or HCO₃ ⁻). Many diseases of mucosal surfaces are caused by toolittle protective liquid on those mucosal surfaces created by animbalance between secretion (too little) and absoption (relatively toomuch). The defective salt transport processes that characterize thesemucosal dysfunctions reside in the epithelial layer of the mucosalsurface.

[0005] One approach to replenish the protective liquid layer on mucosalsurfaces is to “rebalance” the system by blocking Na⁺ channel and liquidabsorption. The epithelial protein that mediates the rate-limiting stepof Na⁺ and liquid absorption is the epithelial Na⁺ channel (ENaC). ENaCis positioned on the apical surface of the epithelium, i.e. the mucosalsurface-environmental interface. Therefore, to inhibit ENaC mediated Na⁺and liquid absorption, an ENaC blocker of the amiloride class (whichblocks from the extracellular domain of ENaC) must be delivered to themucosal surface and, importantly, be maintained at this site, to achievetherapeutic utility. The present invention describes diseasescharacterized by too little liquid on mucosal surfaces and “topical”sodium channel blockers designed to exhibit the increased potency,reduced mucosal abosrption, and slow dissociation (“unbinding” ordetachment) from ENaC required for therapy of these diseases.

[0006] Chronic bronchitis (CB), including the most common lethal geneticform of chronic bronchitis, cystic fibrosis (CF), are diseases thatreflect the body's failure to clear mucus normally from the lungs, whichultimately produces chronic airways infection. In the normal lung, theprimary defense against chronic intrapulmonary airways infection(chronic bronchitis) is mediated by the continuous clearance of mucusfrom bronchial airway surfaces. This function in health effectivelyremoves from the lung potentially noxious toxins and pathogens. Recentdata indicate that the initiating problem, i.e., the “basic defect,” inboth CB and CF is the failure to clear mucus from airway surfaces. Thefailure to clear mucus reflects an imbalance between the amount ofliquid and mucin on airway surfaces. This “airway surface liquid” (ASL)is primarily composed of salt and water in proportions similar to plasma(i.e., isotonic). Mucin macromolecules organize into a well defined“mucus layer” which normally traps inhaled bacteria and is transportedout of the lung via the actions of cilia which beat in a watery, lowviscosity solution termed the “periciliary liquid” (PCL). In the diseasestate, there is an imbalance in the quantities of mucus as ASL on airwaysurfaces. This results in a relative reduction in ASL which leads tomucus concentration, reduction in the lubricant activity of the PCL, anda failure to clear mucus via ciliary activity to the mouth. Thereduction in mechanical clearance of mucus from the lung leads tochronic bacterial colonization of mucus adherent to airway surfaces. Itis the chronic retention of bacteria, the failure of local antimicrobialsubstances to kill mucus-entrapped bacteria on a chronic basis, and theconsequent chronic inflammatory responses of the body to this type ofsurface infection, that lead to the syndromes of CB and CF.

[0007] The current afflicted population in the U.S. is 12,000,000patients with the acquired (primarily from cigarette smoke exposure)form of chronic bronchitis and approximately 30,000 patients with thegenetic form, cystic fibrosis. Approximately equal numbers of bothpopulations are present in Europe. In Asia, there is little CF but theincidence of CB is high and, like the rest of the world, is increasing.

[0008] There is currently a large, unmet medical need for products thatspecifically treat CB and CF at the level of the basic defect that causethese diseases. The current therapies for chronic bronchitis and cysticfibrosis focus on treating the symptoms and/or the late effects of thesediseases. Thus, for chronic bronchitis, β-agonists, inhaled steroids,anti-cholinergic agents, and oral theophyllines and phosphodiesteraseinhibitors are all in development. However, none of these drugs treateffectively the fundamental problem of the failure to clear mucus fromthe lung. Similarly, in cystic fibrosis, the same spectrum ofpharmacologic agents is used. These strategies have been complemented bymore recent strategies designed to clear the CF lung of the DNA(“Pulmozyme”; Genentech) that has been deposited in the lung byneutrophils that have futilely attempted to kill the bacteria that growin adherent mucus masses and through the use of inhaled antibiotics(“TOBI”) designed to augment the lungs' own killing mechanisms to ridthe adherent mucus plaques of bacteria. A general principle of the bodyis that if the initiating lesion is not treated, in this case mucusretention/obstruction, bacterial infections became chronic andincreasingly refractory to antimicrobial therapy. Thus, a major unmettherapeutic need for both CB and CF lung diseases is an effective meansof re-hydrating airway mucus (i.e., restoring/expanding the volume ofthe ASL) and promoting its clearance, with bacteria, from the lung.

[0009] R. C. Boucher, in U.S. Pat. No. 6,264,975, describes the use ofpyrazinoylguanidine sodium channel blockers for hydrating mucosalsurfaces. These compounds, typified by the well-known diureticsamiloride, benzamil, and phenamil, are effective. However, thesecompounds suffer from the significant disadvantage that they are (1)relatively impotent, which is important because the mass of drug thatcan be inhaled by the lung is limited; (2) rapidly absorbed, whichlimits the half-life of the drug on the mucosal surface; and (3) arefreely dissociable from ENaC. The sum of these disadvantages embodied inthese well known diurectics produces compounds with insufficient potencyand/or effective half-life on mucosal surfaces to have therapeuticbenefit for hydrating mucosal surfaces.

[0010] Clearly, what is needed are drugs that are more effective atrestoring the clearance of mucus from the lungs of patients with CB/CF.The value of these new therapies will be reflected in improvements inthe quality and duration of life for both the CF and the CB populations.

[0011] Other mucosal surfaces in and one the body exhibit subtledifferences in the normal physiology of the protective surface liquidson their surfaces but the pathophysiology of disease reflects a commontheme, i.e., too little protective surface liquid. For example, inxerostomia (dry mouth) the oral cavity is depleted of liquid due to afailure of the parotid sublingual and submandibular glands to secreteliquid despite continued Na⁺ (ENaC) transport mediated liquid absorptionfrom the oral cavity. Similarly, keratoconjunctivitis sira (dry eye) iscaused by failure of lacrimal glands to secrete liquid in the face ofcontinued Na⁺ dependent liquid absorption on conjunctional surfaces. Inrhinosinusitis, there is an imbalance, as in CB, between mucin secretionand relative ASL depletion. Finally, in the gastrointestinal tract,failure to secrete Cl⁻ (and liquid) in the proximal small intestine,combined with increased Na⁺ (and liquid) absorption in the terminalileum leads to the distal intestinal obstruction syndrome (DIOS). Inolder patients excessive Na⁺ (and volume) absorption in the descendingcolon produces constipation and diverticulitis.

SUMMARY OF THE INVENTION

[0012] It is an object of the present invention to provide compoundsthat are more potent and/or absorbed less rapidly from mucosal surfaces,and/or are less reversible as compared to known compounds.

[0013] It is another aspect of the present invention to providecompounds of formula (I) that are more potent and/or absorbed lessrapidly and/or exhibit less reversibility, as compared to compounds suchas amilorde, benzamil, and phenamil. Therefore, the compounds of formula(I) will give a prolonged pharmacodynamic half-life on mucosal surfacesas compared to known compounds.

[0014] It is another object of the present invention to providecompounds of formula (I) which are (1) absorbed less rapidly frommucosal surfaces, especially airway surfaces, as compared to knowncompounds and; (2) when absorbed from musosal surfaces afteradministration to the mucosal surfaces, are converted in vivo intometabolic derivitives thereof which have reduced efficacy in blockingsodium channels as compared to the administered parent compound.

[0015] It is another object of the present invention to providecompounds of formula (I) that are more potent and/or absorbed lessrapidly and/or exhibit less reversibility, as compared to compounds suchas amiloride, benzamil, and phenamil. Therefore, the compounds offormula (I) will give a prolonged pharmacodynamic half-life on mucosalsurfaces as compared to previous compounds.

[0016] It is another object of the present invention to provide methodsof treatment which take advantage of the properties described above.

[0017] The objects of the present invention may be accomplished with aclass of pyrazinoylguanidine compounds represented by formula (I):

[0018] where

[0019] X is hydrogen, halogen, trifluoromethyl, lower alkyl,unsubstituted or substituted phenyl, lower alkyl-thio, phenyl-loweralkyl-thio, lower alkyl-sulfonyl, or phenyl-lower alkyl-sulfonyl;

[0020] Y is hydrogen, hydroxyl, mercapto, lower alkoxy, loweralkyl-thio, halogen, lower alkyl, unsubstituted or substitutedmononuclear aryl, or —N(R²)₂;

[0021] R¹ is hydrogen or lower alkyl;

[0022] each R² is, independently, —R⁷, —(CH₂)_(m)—OR⁸,—(CH₂)_(m)—NR⁷R¹⁰, —(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂CH₂)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—(CH₂)_(n)—Z_(g)—R⁷, —(CH₂)_(m)—NR₁₀—CH₂(CHOR⁸)_(n)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂)_(n)—CO₂R⁷, or

[0023] R³ and R⁴ are each, independently, hydrogen, a group representedby formula (A), lower alkyl, hydroxy lower alkyl, phenyl, phenyl-loweralkyl, (halophenyl)-lower alkyl, lower-(alkylphenylalkyl), lower(alkoxyphenyl)-lower alkyl, naphthyl-lower alkyl, or pyridyl-loweralkyl, with the proviso that at least one of R³ and R⁴ is a grouprepresented by formula (A):

[0024]  wherein

[0025] each R^(L) is, independently, —R⁷, —(CH₂)_(n)—OR⁸,—O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)—(Z)_(g)—R⁷,—O—(CH₂)_(m)—(Z)_(g)—R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose

[0026] each o is, independently, an integer from 0 to 10;

[0027] each p is an integer from 0 to 10;

[0028] with the proviso that the sum of o and p in each contiguous chainis from 1 to 10;

[0029] each x is, independently, O, NR¹⁰, C(═O), CHOH, C(═N—R¹⁰),CHNR⁷R¹⁰, or represents a single bond;

[0030] each R⁵ is, independently, —(CH₂)_(m)—OR⁸, —O—(CH₂)_(m)—OR⁸,—(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)—(Z)_(g)—R⁷,—O—(CH₂)_(m)—(Z)_(g)—R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

[0031] each R⁶ is, independently, —R⁷, —OR¹¹, —N(R⁷)₂, —(CH₂)_(m)—OR⁸,—O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)—(Z)_(g)—R⁷,—O—(CH₂)_(m)—(Z)_(g)—R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

[0032] wherein when two R⁶ are —OR¹¹ and are located adjacent to eachother on a phenyl ring, the alkyl moieties of the two R⁶ may be bondedtogether to form a methylenedioxy group;

[0033] each R⁷ is, independently, hydrogen or lower alkyl;

[0034] each R⁸ is, independently, hydrogen, lower alkyl, —C(═O)—R¹¹,glucuronide, 2-tetrahydropyranyl, or

[0035] each R⁹ is, independently, —CO₂R⁷, —CON(R⁷)₂, —SO₂CH₃, or—C(═O)R⁷;

[0036] each R¹⁰ is, independently, —H, —SO₂CH₃, —CO₂R⁷, —C(═O)NR⁷R⁹,—C(═O)R⁷, or —CH₂—(CHOH)_(n)—CH₂OH;

[0037] each Z is, independently, CHOH, C(═O), CHNR⁷R¹⁰, C═NR¹⁰, or NR¹⁰;

[0038] each R¹¹ is, independently, lower alkyl;

[0039] each g is, independently, an integer from 1 to 6;

[0040] each m is, independently, an integer from 1 to 7;

[0041] each n is, independently, an integer from 0 to 7;

[0042] each Q is, independently, C—R⁵, C—R⁶, or a nitrogen atom, whereinat

[0043] most three Q in a ring are nitrogen atoms;

[0044] or a pharmaceutically acceptable salt thereof, and

[0045] inclusive of all enantiomers, diastereomers, and racemic mixturesthereof.

[0046] The present also provides pharmaceutical compositions whichcontain a compound represented above.

[0047] The present invention also provides a method of promotinghydration of mucosal surfaces, comprising:

[0048] administering an effective amount of a compound represented byformula (I) to a mucosal surface of a subject.

[0049] The present invention also provides a method of restoring mucosaldefense, comprising:

[0050] topically administering an effective amount of compoundrepresented by formula (I) to a mucosal surface of a subject in needthereof.

[0051] The present invention also provides a method of blocking ENaC,comprising:

[0052] contacting sodium channels with an effective amount of a compoundrepresented by formula (I).

[0053] The present invention also provides a method of promoting mucusclearance in mucosal surfaces, comprising:

[0054] administering an effective amount of a compound represented byformula (I) to a mucosal surface of a subject.

[0055] The present invention also provides a method of treating chronicbronchitis, comprising:

[0056] administering an effective amount of a compound represented byformula (I) to a subject in need thereof.

[0057] The present invention also provides a method of treating cysticfibrosis, comprising:

[0058] administering an effective amount of compound represented byformula (I) to a subect in need thereof.

[0059] The present invention also provides a method of treatingrhinosinusitis, comprising:

[0060] administering an effective amount of a compound represented by aformula (I) to a subject in need thereof.

[0061] The present invention also provides a method of treating nasaldehydration, comprising:

[0062] administering an effective amount of a compound represented byformula (I) to the nasal passages of a subject in need thereof.

[0063] The present invention also provides a method of treating nasaldehydration, where the nasal dehydration is brought on by administeringdry oxygen to the subject.

[0064] The present invention also provides a method of treatingsinusitis, comprising:

[0065] administering an effective amount of a compound represented byformula (I) to a subject in need thereof.

[0066] The present invention also provides a method of treatingpneumonia, comprising:

[0067] administering an effective amount of a compound represented byformula (I) to a subject in need thereof.

[0068] The present invention also provides a method of preventingventilator-induced pneumonia, comprising:

[0069] admiistering an effective compound represented by formula (I) toa subject by means of a ventilator.

[0070] The present invention also provides a method of treating asthma,comprising:

[0071] administering an effective amount of a compound represented byformula (I) to a subject in need thereof.

[0072] The present invention also provides a method of treating primaryciliary dyskinesia, comprising:

[0073] administering an effective amount of a compound represented byformula (I) to a subject in need thereof.

[0074] The present invention also provides a method of treating otitismedia, comprising:

[0075] administering an effective amount of a compound represented byformula (I) to a subject in need thereof.

[0076] The present invention also provides a method of inducing sputumfor diagnostic purposes, comprising:

[0077] administering an effective amount of compound represented byformula (I) to a subject in need thereof.

[0078] The present invention also provides a method of treating chronicobstructive pulmonary disease, comprising:

[0079] administering an effective amount of a compound represented byformula (I) to a subject in need thereof.

[0080] The present invention also provides a method of treatingemphysema, comprising:

[0081] administering an effective amount of a compound represented byformula (I) to a subject in need thereof.

[0082] The present invention also provides a method of treating dry eye,comprising:

[0083] administering an effective amount of a compound represented byformula (I) to the eye of the subject in need thereof.

[0084] The present invention also provides a method of promoting ocularhydration, comprising:

[0085] administering an effective amount of a compound represented byformula (I) to the eye of the subject.

[0086] The present invention also provides a method of promoting cornealhydration, comprising:

[0087] administering an effective amount of a compound represented byformula (I) to the eye of the subject.

[0088] The present invention also provides a method of treatingSjogren's disease, comprising:

[0089] administering an effective amount of compound represented byformula (I) to a subject in need thereof.

[0090] The present invention also provides a method of treating vaginaldryness, comprising:

[0091] administering an effective amount of a compound represented byformula (I) to the vaginal tract of a subject in need thereof.

[0092] The present invention also provides a method of treating dryskin, comprising:

[0093] administering an effective amount of a compound represented byformula (I) to the skin of a subject in need thereof.

[0094] The present invention also provides a method of treating drymouth (xerostomia), comprising:

[0095] administering an effective amount of compound represented byformula (I) to the mouth of the subject in need thereof.

[0096] The present invention also provides a method of treating distalintestinal obstruction syndrome, comprising:

[0097] administering an effective amount of compound represented byformula (I) to a subject in need thereof.

[0098] The present invention also provides a method of treatingesophagitis, comprising:

[0099] administering an effective amount of a compound represented byformula (I) to a subject in need thereof.

[0100] The present invention also provides a method of treatingconstipation, comprising:

[0101] administering an effective amount of a compound represented byformula (I) to a subject in need thereof. In one embodiment of thismethod, the compound is administered either orally or via a suppositoryor enema.

[0102] The present invention also provides a method of treating chronicdiverticulitis comprising:

[0103] administering an effective amount of a compound represented byformula (I) to a subject in need thereof.

BRIEF DESCRIPTION OF THE FIGURES

[0104] A more complete appreciation of the invention and many of theattendant advantages thereof will be readily obtained as the samebecomes better understood by reference to the following detaileddescription considered in conjunction with the following figures:

[0105]FIG. 1: Effect of a compound of the present invention on MCC att=0 hrs as described in Example 32 herein.

[0106]FIG. 2: Effect of a compound of the present invention on MCC att=4 hrs as described in Example 32 herein.

DETAILED DESCRIPTION OF THE INVENTION

[0107] The present invention is based on the discovery that thecompounds of formula (I) are more potent and/or, absorbed less rapidlyfrom mucosal surfaces, especially airway surfaces, and/or lessreversible from interactions with ENaC as compared to compounds such asamiloride, benzamil, and phenamil. Therefore, the compounds of formula(I) have a longer half-life on mucosal surfaces as compared to thesecompounds.

[0108] The present invention is also based on the discovery that certaincompounds embraced by formula (I) are converted in vivo into metabolicderivitives thereof that have reduced efficacy in blocking sodiumchannels as compared to the parent administered compound, after they areabsorbed from musosal surfaces after administration. This importantproperty means that the compounds will have a lower tendency to causeundesired side-effects by blocking sodium channels located at untargetedlocations in the body of the recipient, e.g., in the kidneys.

[0109] In the compounds represented by formula (I), X may be hydrogen,halogen, trifluoromethyl, lower alkyl, lower cycloalkyl, unsubstitutedor substituted phenyl, lower alkyl-thio, phenyl-lower alkyl-thio, loweralkyl-sulfonyl, or phenyl-lower alkyl-sulfonyl. Halogen is preferred.

[0110] Examples of halogen include fluorine, chlorine, bromine, andiodine. Chlorine and bromine are the preferred halogens. Chlorine isparticularly preferred. This description is applicable to the term“halogen” as used throughout the present disclosure.

[0111] As used herein, the term “lower alkyl” means an alkyl grouphaving less than 8 carbon atoms. This range includes all specific valuesof carbon atoms and subranges there between, such as 1 ,2, 3, 4, 5, 6,and 7 carbon atoms. The term “alkyl” embraces all types of such groups,e.g., linear, branched, and cyclic alkyl groups. This description isapplicable to the term “lower alkyl” as used throughout the presentdisclosure. Examples of suitable lower alkyl groups include methyl,ethyl, propyl, cyclopropyl, butyl, isobutyl, etc.

[0112] Substituents for the phenyl group include halogens. Particularlypreferred halogen substituents are chlorine and bromine.

[0113] Y may be hydrogen, hydroxyl, mercapto, lower alkoxy, loweralkyl-thio, halogen, lower alkyl, lower cycloalkyl, mononuclear aryl, or—N(R²)₂. The alkyl moiety of the lower alkoxy groups is the same asdescribed above. Examples of mononuclear aryl include phenyl groups. Thephenyl group may be unsubstituted or substituted as described above. Thepreferred identity of Y is —N(R²)₂. Particularly preferred are suchcompounds where each R² is hydrogen.

[0114] R¹ may be hydrogen or lower alkyl. Hydrogen is preferred for R¹.

[0115] Each R² may be, independently, —R⁷, —(CH₂)_(m)—OR⁸,—(CH₂)_(m)—NR⁷R¹⁰, —(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—(CH₂)_(n)—Z_(g)—R⁷, —(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂)_(n)—CO₂R⁷, or

[0116] Hydrogen and lower alkyl, particularly C₁-C₃ alkyl are preferredfor R². Hydrogen is particularly preferred.

[0117] R³ and R⁴ may be, independently, hydrogen, a group represented byformula (A), lower alkyl, hydroxy lower alkyl, phenyl, phenyl-loweralkyl, (halophenyl)-lower alkyl, lower-(alkylphenylalkyl), lower(alkoxyphenyl)-lower alkyl, naphthyl-lower alkyl, or pyridyl- loweralkyl, provided that at least one of R³ and R⁴ is a group represented byformula (A).

[0118] Preferred compounds are those where one of R³ and R⁴ is hydrogenand the other is represented by formula (A).

[0119] In formula (A), the moiety —(C(R^(L))₂)_(o)-x-(C(R^(L))₂)_(p)—defines an alkylene group bonded to the aromatic ring. The variables oand p may each be an integer from 0 to 10, subject to the proviso thatthe sum of o and p in the chain is from 1 to 10. Thus, o and p may eachbe 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Preferably, the sum of o and pis from 2 to 6. In a particularly preferred embodiment, the sum of o andp is 4.

[0120] The linking group in the alkylene chain, x, may be,independently, O, NR¹⁰, C(═O), CHOH, C(═N—R¹⁰), CHNR⁷R¹⁰, or representsa single bond;

[0121] Therefore, when x represents a single bond, the alkylene chainbonded to the ring is represented by the formula —(C(R^(L))₂)_(0+p)—, inwhich the sum o+p is from 1 to 10.

[0122] Each R^(L) may be, independently, —R⁷, —(CH₂)_(n)—OR⁸,—O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)—(Z)_(g)—R⁷,—O—(CH₂)_(m)—(Z)_(g)—R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

[0123] The preferred R^(L) groups include —H, —OH, —N(R⁷)₂, especiallywhere each R⁷ is hydrogen.

[0124] In the alkylene chain in formula (A), it is preferred that whenone R^(L) group bonded to a carbon atoms is other than hydrogen, thenthe other R^(L) bonded to that carbon atom is hydrogen, i.e., theformula —CHR^(L)—. It is also preferred that at most two R^(L) groups inan alkylene chain are other than hydrogen, where in the other R^(L)groups in the chain are hydrogens. Even more preferably, only one R^(L)group in an alkylene chain is other than hydrogen, where in the otherR^(L) groups in the chain are hydrogens. In these embodiments, it ispreferable that x represents a single bond.

[0125] In another particular embodiment of the invention, all of theR^(L) groups in the alkylene chain are hydrogen. In these embodiments,the alkylene chain is represented by the formula (CH₂)_(o)-x-(CH₂)_(p)—.

[0126] Each R⁵ may be, independently, —(CH₂)_(m)—OR⁸, —O—(CH₂)_(m)—OR⁸,—(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)—(Z)_(g)—R⁷,—O—(CH₂)_(m)—(Z)_(g)—R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(m)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(m)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

[0127] There are four R⁶ groups present on the ring in formula (A). EachR⁶ may be each, independently, —R⁷, —OR¹¹, —N(R⁷)₂, —(CH₂)_(m)—R⁸,—O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)—(Z)_(g)—R⁷,—O—(CH₂)_(m)—(Z)_(g)—R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose, or

[0128] When two R⁶ are —OR¹¹ and are located adjacent to each other on aphenyl ring, the alkyl moieties of the two R⁶ groups may be bondedtogether to form a methylenedioxy group, i.e., a group of the formula—O—CH₂—O—.

[0129] As discussed above, R⁶ may be hydrogen. Therefore, 1, 2, 3, or 4R⁶ groups may be other than hydrogen. Preferably at most 3 of the R⁶groups are other than hydrogen.

[0130] Each g is, independently, an integer from 1 to 6. Therefore, eachg may be 1, 2, 3, 4, 5, or 6.

[0131] Each m is an integer from 1 to 7. Therefore, each m may be 1, 2,3, 4, 5, 6, or 7.

[0132] Each n is an integer from 0 to 7. Therefore, each n maybe 0, 1,2, 3, 4, 5, 6, or 7.

[0133] Each Q in formula (A) is C—R⁵, C—R⁶, or a nitrogen atom, where atmost three Q in a ring are nitrogen atoms. Thus, there may be 1, 2, or 3nitrogen atoms in a ring. Preferably, at most two Q are nitrogen atoms.More preferably, at most one Q is a nitrogen atom. In one particularembodiment, the nitrogen atom is at the 3-position of the ring. Inanother embodiment of the invention, each Q is either C—R⁵ or C—R⁶,i.e., there are no nitrogen atoms in the ring.

[0134] More specific examples of suitable groups represented by formula(A) are shown in formulas (B)-(F) below:

[0135] where o, x, p, R⁵, and R⁶, are as defined above;

[0136] where n is an integer from 1 to 10 and R⁵ is as defined above;

[0137] where n is an integer from 1 from 10 and R⁵ is as defined above;

[0138] where o, x, p, and R⁵ are as defined above;

[0139] where n and m are as defined above.

[0140] In a preferred embodiment of the invention, Y is —NH₂.

[0141] In another preferred embodiment, R² is hydrogen.

[0142] In another preferred embodiment, R¹ is hydrogen.

[0143] In another preferred embodiment, X is chlorine.

[0144] In another preferred embodiment, R³ is hydrogen.

[0145] In another preferred embodiment, R^(L) is hydrogen.

[0146] In another preferred embodiment, o is 4.

[0147] In another preferred embodiment, p is 0.

[0148] In another preferred embodiment, the sum of 0 and p is 4.

[0149] In another preferred embodiment, x represents a single bond.

[0150] In another preferred embodiment, R⁶ is hydrogen.

[0151] In another preferred embodiment, at most one Q is a nitrogenatom.

[0152] In another preferred embodiment, no Q is a nitrogen atom.

[0153] In a preferred embodiment of the present invention:

[0154] X is halogen;

[0155] Y is —N(R⁷)₂;

[0156] R¹ is hydrogen or C₁-C₃ alkyl;

[0157] R² is —R⁷, —OR⁷, CH₂O⁷, or —CO₂R⁷;

[0158] R³ is a group represented by formula (A); and

[0159] R⁴ is hydrogen, a group represented by formula (A), or loweralkyl;

[0160] In another preferred embodiment of the present invention:

[0161] X is chloro or bromo;

[0162] Y is —N(R⁷)₂;

[0163] R² is hydrogen or C₁-C₃ alkyl;

[0164] at most three R⁶ are other than hydrogen as described above;

[0165] at most three R^(L) are other than hydrogen as described above;and

[0166] at most 2 Q are nitrogen atoms.

[0167] In another preferred embodiment of the present invention:

[0168] Y is —NH₂;

[0169] In another preferred embodiment of the present invention:

[0170] R⁴ is hydrogen;

[0171] at most one R^(L) is other than hydrogen as described above;

[0172] at most two R⁶ are other than hydrogen as described above; and

[0173] at most 1 Q is a nitrogen atom.

[0174] In another preferred embodiment of the present invention R⁵ isselected from the group consisting of

[0175] —O—(CH₂)₃—OH, —NH₂, —O—CH₂—(CHOH)₂—CH₂OH, —O—CH₂—CHOH—CH₂OH,—O—CH₂CH₂—O-tetrahydropyran-2-yl, —O—CH₂CHOH—CH₂—O-glucuronide,—O—CH₂CH₂OH, —O—(CH₂CH₂O)₄—CH₃, —O—CH₂CH₂OCH₃,—O—CH₂—(CHOC(═O)CH₃)—CH₂—OC(═O)CH₃, —O—(CH₂CH₂O)₂—CH₃,—OCH₂—CHOH—CHOH—CH₂OH, —CH₂OH, —CO₂CH₃, and

[0176] In another preferred embodiment, R⁵ is selected from the groupconsisting of para —O—(CH₂)₃—OH, para —NH₂, para —O—CH₂—(CHOH)₂—CH₂OH,para —O—CH₂CH₂—O—tetrahydropyran-2-yl, para—O—CH₂CHOH—CH₂—O-glucuronide, para —O—CH₂CH₂OH, para —O—(CH₂CH₂O)₄—CH₃,para —O—CH₂CH₂OCH₃, para —O—CH₂—(CHOC(═O)CH₃)—CH₂—OC(═O)CH₃, para—O—(CH₂CH₂O)₂—CH₃, —OCH₂—CHOH—CHOH—CH₂OH, para —CH₂OH, para —CO₂CH₃,para —SO₃H, para —O-glucuronide, para

[0177] In another particularly preferred embodiment of the invention,the compound of formula (I) is represented by the formula (Ia), (Ib),(Ic), or (Id):

[0178] or a pharmaceutically acceptable salt thereof.

[0179] The compounds of formula (I) may be prepared and used as the freebase. Alternatively, the compounds may be prepared and used as apharmaceutically acceptable salt. Pharmaceutically acceptable salts aresalts that retain or enhance the desired biological activity of theparent compound and do not impart undesired toxicological effects.Examples of such salts are (a) acid addition salts formed with inorganicacids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid and the like; (b) salts formed with organicacids such as, for example, acetic acid, oxalic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonicacid, polygalacturonic acid, malonic acid, sulfosalicylic acid, glycolicacid, 2-hydroxy-3-naphthoate, pamoate, salicylic acid, stearic acid,phthalic acid, mandelic acid, lactic acid and the like; and (c) saltsformed from elemental anions for example,chlorine, bromine, and iodine.

[0180] It is to be noted that all enantiomers, diastereomers, andracemic mixtures of compounds within the scope of formula (I) areembraced by the present invention. All mixtures of such enantiomers anddiastereomers are within the scope of the present invention.

[0181] Without being limited to any particular theory, it is believedthat the compounds of formula (I) function in vivo as sodium channelblockers. By blocking epithelial sodium channels present in mucosalsurfaces the compounds of formula (I) reduce the absorption of water bythe mucosal surfaces. This effect increases the volume of protectiveliquids on mucosal surfaces, rebalances the system, and thus treatsdisease.

[0182] The present invention also provides methods of treatment thattake advantage of the properties of the compounds of formula (I)discussed above. Thus, subjects that may be treated by the methods ofthe present invention include, but are not limited to, patients jinxafflicted with cystic fibrosis, primary ciliary dyskinesia, chronicbronchitis, chronic obstructive airway disease, artificially ventilatedpatients, patients with acute pneumonia, etc. The present invention maybe used to obtain a sputum sample from a patient by administering theactive compounds to at least one lung of a patient, and then inducing orcollecting a sputum sample from that patient. Typically, the inventionwill be administered to respiratory mucosal surfaces via aerosol (liquidor dry powders) or lavage.

[0183] Subjects that may be treated by the method of the presentinvention also include patients being administered supplemental oxygennasally (a regimen that tends to dry the airway surfaces); patientsafflicted with an allergic disease or response (e.g., an allergicresponse to pollen, dust, animal hair or particles, insects or insectparticles, etc.) that affects nasal airway surfaces; patients afflictedwith a bacterial infection e.g., staphylococcus infections such asStaphylococcus aureus infections, Hemophilus influenza infections,Streptococcus pneumoniae infections, Pseudomonas aeuriginosa infections,etc.) of the nasal airway surfaces; patients afflicted with aninflammatory disease that affects nasal airway surfaces; or patientsafflicted with sinusitis (wherein the active agent or agents areadministered to promote drainage of congested mucous secretions in thesinuses by administering an amount effective to promote drainage ofcongested fluid in the sinuses), or combined, Rhinosinusitis. Theinvention may be administered to rhino-sinal surfaces by topicaldelivery, including aerosols and drops.

[0184] The present invention may be used to hydrate mucosal surfacesother than airway surfaces. Such other mucosal surfaces includegastrointestinal surfaces, oral surfaces, genito-urethral surfaces,ocular surfaces or surfaces of the eye, the inner ear and the middleear. For example, the active compounds of the present invention may beadministered by any suitable means, including locally/topically, orally,or rectally, in an effective amount.

[0185] The present invention is concerned primarily with the treatmentof human subjects, but may also be employed for the treatment of othermammalian subjects, such as dogs and cats, for veterinary purposes.

[0186] As discussed above, the compounds used to prepare thecompositions of the present invention may be in the form of apharmaceutically acceptable free base. Because the free base of thecompound is generally less soluble in aqueos solutions than the salt,free base compositions are employed to provide more sustained release ofactive agent to the lungs. An active agent present in the lungs inparticulate form which has not dissolved into solution is not availableto induce a physiological response, but serves as a depot ofbioavailable drug which gradually dissolves into solution.

[0187] Another aspect of the present invention is a pharmaceuticalcomposition, comprising a compound of formula (I) in a pharmaceuticallyacceptable carrier (e.g., an aqueous carrier solution). In general, thecompound of formula (I) is included in the composition in an amounteffective to inhibit the reabsorption of water by mucosal surfaces.

[0188] The compounds of the present invention may also be used inconjunction with a P2Y2 (receptor agonist or a pharmaceuticallyacceptable salt thereof (also sometimes referred to as an “active agent”herein). The composition may further comprise a P2Y2 receptor agonist ora pharmaceutically acceptable salt thereof (also sometimes referred toas an “active agent” herein). The P2Y2 receptor agonist is typicallyincluded in an amount effective to stimulate chloride and watersecretion by airway surfaces, particularly nasal airway surfaces.Suitable P2Y2 receptor agonists are described in columns 9-10 of U.S.Pat. No. 6,264,975, U.S. Pat. No. 5,656,256, and U.S. Pat. No.5,292,498, each of which is incorporated herein by reference.

[0189] Bronchodiloators can also be used in combination with compoundsof the present invention. These Bronchodilators include, but are notlimited to, β-adrenergic agonists including but not limited toepinephrine, isoproterenol, fenoterol, albutereol, terbutalin,pirbuterol, bitolterol, metaproterenol, iosetharine, salmeterolxinafoate, as well as anticholinergic agents including but not limitedto ipratropium bromide, as well as compounds such as theophylline andaminophylline. These compounds may be administered in accordance withknown techniques, either prior to or concurrently with the activecompounds described herein.

[0190] Another aspect of the present invention is a pharmaceuticalformulation, comprising an active compound as described above in apharmaceutically acceptable carrier (e.g., an aqueous carrier solution).In general, the active compound is included in the composition in anamount effective to treat mucosal surfaces, such as inhibiting thereabsorption of water by mucosal surfaces, including airway and othersurfaces.

[0191] The active compounds disclosed herein may be administered tomucosal surfaces by any suitable means, including topically, orally,rectally, vaginally, ocularly and dermally, etc. For example, for thetreatment of constipation, the active compounds may be administeredorally or rectally to the gastrointestinal mucosal surface. The activecompound may be combined with a pharmaceutically acceptable carrier inany suitable form, such as sterile physiological or dilute saline ortopical solution, as a droplet, tablet or the like for oraladministration, as a suppository for rectal or genito-urethraladministration, etc. Excipients may be included in the formulation toenhance the solubility of the active compounds, as desired.

[0192] The active compounds disclosed herein may be administered to theairway surfaces of a patient by any suitable means, including as aspray, mist, or droplets of the active compounds in a pharmaceuticallyacceptable carrier such as physiological or dilute saline solutions ordistilled water. For example, the active compounds may be prepared asformulations and administered as described in U.S. Pat. No. 5,789,391 toJacobus, the disclosure of which is incorporated by reference herein inits entirety.

[0193] Solid or liquid particulate active agents prepared for practicingthe present invention could, as noted above, include particles ofrespirable or non-respirable size; that is, for respirable particles,particles of a size sufficiently small to pass through the mouth andlarynx upon inhalation and into the bronchi and alveoli of the lungs,and for non-respirable particles, particles sufficiently large to beretained in the nasal airway passages rather than pass through thelarynx and into the bronchi and alveoli of the lungs. In general,particles ranging from about 1 to 5 microns in size (more particularly,less than about 4.7 microns in size) are respirable. Particles ofnon-respirable size are greater than about 5 microns in size, up to thesize of visible droplets. Thus, for nasal administration, a particlesize in the range of 10-500 μm may be used to ensure retention in thenasal cavity.

[0194] In the manufacture of a formulation according to the invention,active agents or the physiologically acceptable salts or free basesthereof are typically admixed with, inter alia, an acceptable carrier.Of course, the carrier must be compatible with any other ingredients inthe formulation and must not be deleterious to the patient. The carriermust be solid or liquid, or both, and is preferably formulated with thecompound as a unit-dose formulation, for example, a capsule, that maycontain 0.5% to 99% by weight of the active compound.

[0195] One or more active compounds may be incorporated in theformulations of the invention, which formulations may be prepared by anyof the well-known techniques of pharmacy consisting essentially ofadmixing the components.

[0196] Compositions containing respirable or non-respirable dryparticles of micronized active agent may be prepared by grinding the dryactive agent with a mortar and pestle, and then passing the micronizedcomposition through a 400 mesh screen to break up or separate out largeagglomerates.

[0197] The particulate active agent composition may optionally contain adispersant which serves to facilitate the formulation of an aerosol. Asuitable dispersant is lactose, which may be blended with the activeagent in any suitable ratio (e.g., a 1 to 1 ratio by weight).

[0198] Active compounds disclosed herein may be administered to airwaysurfaces including the nasal passages, sinuses and lungs of a subject byan suitable means know in the art, such as by nose drops, mists., etc.In one embodiment of the invention, the active compounds of the presentinvention and administered by transbronchoscopic lavage. In a preferredembodiment of the invention, the active compounds of the presentinvention are deposited on lung airway surfaces by administering anaerosol suspension of respirable particles comprised of the activecompound, which the subject inhales. The respirable particles may beliquid or solid. Numerous inhalers for administering aerosol particlesto the lungs of a subject are known.

[0199] Inhalers such as those developed by Inhale Therapeutic Systems,Palo Alto, Calif., USA, may be employed, including but not limited tothose disclosed in U.S. Pat. Nos. 5,740,794; 5,654,007; 5,458,135;5,775,320; and 5,785,049. The Applicant specifically intends that thedisclosures of all patent references cited herein be incorporated byreference herein in their entirety. Inhalers such as those developed byDura Pharmaceuticals, Inc., San Diego, Calif., USA, may also beemployed, including but not limited to those disclosed in U.S. Pat. Nos.5,622,166; 5,577,497; 5,645,051; and 5,492,112. Additionally, inhalerssuch as those developed by Aradigm Corp., Hayward, Calif., USA, may beemployed, including but not limited to those disclosed in U.S. Pat. Nos.5,826,570; 5,813,397; 5,819,726; and 5,655,516. These apparatuses areparticularly suitable as dry particle inhalers.

[0200] Aerosols of liquid particles comprising the active compound maybe produced by any suitable means, such as with a pressure-drivenaerosol nebulizer or an ultrasonic nebulizer. See, e.g., U.S. Pat. No.4,501,729. Nebulizers are commercially available devices which transformsolutions or suspensions of the active ingredient into a therapeuticaerosol mist either by means of acceleration of compressed gas,typically air or oxygen, through a narrow venturi orifice or by means ofultrasonic agitation. Suitable formulations for use in nebulizersconsist of the active ingredient in a liquid carrier, the activeingredient comprising up to 40% w/w of the formulation, but preferablyless than 20% w/w. The carrier is typically water (and most preferablysterile, pyrogen-free water) or dilute aqueous alcoholic solution.Perfluorocarbon carriers may also be used. Optional additives includepreservatives if the formulation is not made sterile, for example,methyl hydroxybenzoate, antioxidants, flavoring agents, volatile oils,buffering agents and surfactants.

[0201] Aerosols of solid particles comprising the active compound maylikewise be produced with any solid particulate medicament aerosolgenerator. Aerosol generators for administering solid particulatemedicaments to a subject produce particles which are respirable, asexplained above, and generate a volume of aerosol containingpredetermined metered dose of medicament at a rate suitable for humanadministration. One illustrative type of solid particulate aerosolgenerator is an insufflator. Suitable formulations for administration byinsufflation include finely comminuted powders which may be delivered bymeans of an insufflator or taken into the nasal cavity in the manner ofa snuff. In the insufflator, the powder (e.g., a metered dose thereofeffective to carry out the treatments described herein) is contained incapsules or cartridges, typically made of gelatin or plastic, which areeither pierced or opened in situand the powder delivered by air drawnthrough the device upon inhalation or by means of a manually-operatedpump. The powder employed in the insufflator consists either solely ofthe active ingredient or of powder blend comprising the activeingredient, a suitable powder diluent, such as lactose, and an optionalsurfactant. The active ingredient typically comprises of 0.1 to 100% w/wof the formulation. A second type of illustrative aerosol generatorcomprises a metered dose inhaler. Metered dose inhalers are pressurizedaerosol dispensers, typically containing a suspension or solutionformulation of active ingredient in a liquified propellant. During use,these devices discharge the formulation through a valve adapted todeliver a metered volume, typically from 10 to 150 μl, to produce a fineparticle spray containing the active ingredient. Suitable propellantsinclude certain chlorofluorocarbon compounds, for example,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane and mixtures thereof. The formulation mayadditionally contain one of more co-solvents, for example, ethanol,surfactants, such as oleic acid or sorbitan trioleate, antioxidants andsuitable flavoring agents.

[0202] The aerosol, whether formed from solid or liquid particles, maybe produced by the aerosol generator at a rate of from about 10 to 150liters per minute, more preferable from 30 to 150 liters per minute, andmost preferably about 60 liters per minute. Aerosols containing greateramounts of medicament may be administered more rapidly.

[0203] The dosage of the active compounds disclosed herein will varydepending on the condition being treated and the state of the subject,but generally may be from about 0.01, 0.03, 0.05, 0.1 to 1, 5, 10 or 20mg of the pharmaceutic agent, deposited on the airway surfaces. Thedaily dose may be divided among one or multiple unit doseadministrations. The goal is to achieve a concentration of thepharmaceutic agents on lung airway surfaces of between 10⁻⁹-10⁴ M.

[0204] In another embodiment, they are administered by administering anaerosol suspension of respirable or non-respirable particles (preferablynon-respirable particles) comprised of active compound, which thesubject inhales through the nose. The respirable or non-respirableparticles may be liquid or solid. The quantity of active agent includedmay be an amount of sufficient to achieve dissolved concentrations ofactive agent on the airway surfaces of the subject of from about 10⁻⁹,10⁻⁸, or 10⁻⁷ to about 10⁻³, 10⁻², 10⁻¹ Moles/liter, and more preferablyfrom about 10⁻⁹ to about 10⁻⁴ Moles/liter.

[0205] The dosage of active compound will vary depending on thecondition being treated and the state of the subject, but generally maybe an amount sufficient to achieve dissolved concentrations of activecompound on the nasal airway surfaces of the subject from about 10⁻⁹,10⁻⁸, 10⁻⁷ to about 10⁻³, 10⁻², or 10⁻¹ Moles/liter, and more preferablyfrom about 10⁻⁷ to about 10⁻⁴ Moles/liter. Depending upon the solubilityof the particular formulation of active compound administered, the dailydose may be divided among one or several unit dose administrations. Thedaily dose by weight may range from about 0.01, 0.03, 0.1, 0.5 or 1.0 to10 or 20 milligrams of active agent particles for a human subject,depending upon the age and condition of the subject. A currentlypreferred unit dose is about 0.5 milligrams of active agent given at aregimen of 2-10 administrations per day. The dosage may be provided as aprepackaged unit by any suitable means (e.g., encapsulating a gelatincapsule).

[0206] In one embodiment of the invention, the particulate active agentcomposition may contain both a free base of active agent and apharmaceutically acceptable salt to provide both early release andsustained release of active agent for dissolution into the mucussecretions of the nose. Such a composition serves to provide both earlyrelief to the patient, and sustained relief over time. Sustained relief,by decreasing the number of daily administrations required, is expectedto increase patient compliance with course of active agent treatments.

[0207] Pharmaceutical formulations suitable for airway administrationinclude formulations of solutions, emulsions, suspensions and extracts.See generally, J. Naim, Solutions, Emulsions, Suspensions and Extracts,in Remington: The Science and Practice of Pharmacy, chap. 86 (19^(th) ed1995). Pharmaceutical formulations suitable for nasal administration maybe prepared as described in U.S. Pat. Nos. 4,389,393 to Schor; 5,707,644to Illum; 4,294,829 to Suzuki; and 4,835,142 to Suzuki; the disclosuresof which are incorporated by reference herein in the entirety.

[0208] Mists or aerosols of liquid particles comprising the activecompound may be produced by any suitable means, such as by a simplenasal spray with the active agent in an aqueous pharmaceuticallyacceptable carrier, such as a sterile saline solution or sterile water.Administration may be with a pressure-driven aerosol nebulizer or anultrasonic nebulizer. See e.g. U.S. Pat. Nos. 4,501,729 and 5,656,256.Suitable formulations for use in a nasal droplet or spray bottle or innebulizers consist of the active ingredient in a liquid carrier, theactive ingredient comprising up to 40% w/w of the formulation, butpreferably less than 20% w/w. Typically the carrier is water (and mostpreferably sterile, pyrogen-free water) or dilute aqueous alcoholicsolution, preferably made in a 0.12% to 0.8% solution of sodiumchloride. Optional additives include preservatives if the formulation isnot made sterile, for example, methyl hydroxybenzoate, antioxidants,flavoring agents, volatile oils, buffering agents, osmotically activeagents (e.g. mannitol, xylitol, erythritol) and surfactants.

[0209] Compositions containing respirable or non-respirable dryparticles of micronized active agent may be prepared by grinding the dryactive agent with a mortar and pestle, and then passing the micronizedcomposition through a 400 mesh screen to break up or separate out largeagglomerates.

[0210] The particulate composition may optionally contain a dispersantwhich serves to facilitate the formation of an aerosol. A suitabledispersant is lactose, which may be blended with the active agent in anysuitable ratio (e.g., a 1 to 1 ratio by weight).

[0211] The compounds of formula (I) may be synthesized according toprocedures known in the art. A representative synthetic procedure isshown in the scheme below.

[0212] These procedures are described in, for example, E. J. Cragoe,“The Synthesis of Amiloride and Its Analogs” (Chapter 3) in Amilorideand Its Analogs, pp. 25-36, incorporated herein by reference. Othermethods of preparing the compounds are described in, for example, U.S.Pat. No. 3,313,813, incorporated herein by reference. See in particularMethods A, B, C, and D described in U.S. Pat. No. 3,313,813.

[0213] Several assays may be used to characterize the compounds of thepresent invention. Representative assays are discussed below.

In Vitro Measure of Sodium Channel Blocking Activity and Reversibility

[0214] One assay used to assess mechanism of action and/or potency ofthe compounds of the present invention involves the determination oflumenal drug inhibition of airway epithelial sodium currents measuredunder short circuit current (I_(SC)) using airway epithelial monolayersmounted in Using chambers. Cells obtained from freshly excised human,dog, sheep or rodent airways are seeded onto porous 0.4 micron Snapwell™Inserts (CoStar), cultured at air-liquid interface (ALI) conditions inhormonally defined media, and assayed for sodium transport activity(I_(SC)) while bathed in Krebs Bicarbonate Ringer (KBR) in Usingchambers. All test drug additions are to the lumenal bath with half-logdose addition protocols (from 1×10⁻¹¹ M to 3×10⁻⁵M), and the cumulativechange in Isc (inhibition) recorded. All drugs are prepared in dimethylsulfoxide as stock solutions at a concentration of 1×10⁻² M and storedat −20° C. Eight preparations are typically run in parallel; twopreparations per run incorporate amiloride and/or benzamil as positivecontrols. After the maximal concentration (5×10⁻⁵ M) is administered,the lumenal bath is exchanged three times with fresh drug-free KBRsolution, and the resultant I_(SC) measured after each wash forapproximately 5 minutes in duration. Reversibility is defined as thepercent return to the baseline value for sodium current after the thirdwash. All data from the voltage clamps are collected via a computerinterface and analyzed off-line.

[0215] Dose-effect relationships for all compounds are considered andanalyzed by the Prism 3.0 program. IC₅₀ values, maximal effectiveconcentrations, and reversibility are calculated and compared toamiloride and benzamil as positive controls.

Pharmacological Assays of Absorption

[0216] (1) Apical Disappearance Assay

[0217] Bronchial cells (dog, human, sheep, or rodent cells) are seededat a density of 0.25×10⁶/cm² on a porous Transwell-Col collagen-coatedmembrane with a growth area of 1.13 cm² grown at an air-liquid interfacein hormonally defined media that promotes a polarized epithelium. From12 to 20 days after development of an air-liquid interface (ALI) thecultures are expected to be >90% ciliated, and mucins will accumulate onthe cells. To ensure the integrity of primary airway epithelial cellpreparations, the transepithelial resistance (R_(t)) and transepithelialpotential differences (PD), which are indicators of the integrity ofpolarized nature of the culture, are measured. Human cell systems arepreferred for studies of rates of absorption from apical surfaces. Thedisappearance assay is conducted under conditions that mimic the “thin”films in vivo (˜25 μl) and is initiated by adding experimental sodiumchannel blockers or positive controls (amiloride, benzamil, phenamil) tothe apical surface at an initial concentration of 10 μM. A series ofsamples (5 μl volume per sample) is collected at various time points,including 0, 5, 20, 40, 90 and 240 minutes. Concentrations aredetermined by measuring intrinsic fluorescence of each sodium channelblocker using a Fluorocount Microplate Flourometer or HPLC. Quantitativeanalysis employs a standard curve generated from authentic referencestandard materials of known concentration and purity. Data analysis ofthe rate of disappearance is performed using nonlinear regression, onephase exponential decay (Prism V 3.0).

[0218] 2. Confocal Microscopy Assay of Amiloride Congener Uptake

[0219] Virtually all amiloride-like molecules fluoresce in theultraviolet range. This property of these molecules may be used todirectly measure cellular update using x-z confocal microscopy.Equimolar concentrations of experimental compounds and positive controlsincluding amiloride and compounds that demonstrate rapid uptake into thecellular compartment (benzamil and phenamil) are placed on the apicalsurface of airway cultures on the stage of the confocal microscope.Serial x-z images are obtained with time and the magnitude offluorescence accumulating in the cellular compartment is quantitated andplotted as a change in fluorescence versus time.

[0220] 3. In Vitro Assays of Compound Metabolism

[0221] Airway epithelial cells have the capacity to metabolize drugsduring the process of transepithelial absorption. Further, although lesslikely, it is possible that drugs can be metabolized on airwayepithelial surfaces by specific ectoenzyme activities. Perhaps morelikely as an ecto-surface event, compounds may be metabolized by theinfected secretions that occupy the airway lumens of patients with lungdisease, e.g. cystic fibrosis. Thus, a series of assays is performed tocharacterize the compound metabolism that results from the interactionof test compounds with human airway epithelia and/or human airwayepithelial lumenal products.

[0222] In the first series of assays, the interaction of test compoundsin KBR as an “ASL” stimulant are applied to the apical surface of humanariway epithelial cells grown in the T-Col insert system. For mostcompounds, we test for metabolism (generation of new species) using highperformance liquid chromatography (HPLC) to resolve chemical species andthe endogenous fluorescence properties of these compounds to estimatethe relative quantities of test compound and novel metabolites. For atypical assay, a test solution (25 μl KBR, containing 10 μM testcompound) is placed on the epithelial lumenal surface. Sequential 5 to10 μl samples are obtained from the lumenal and serosal compartments forHPLC analysis of (1) the mass of test compound permeating from thelumenal to serosal bath and (2) the potential formation of metabolitesfrom the parent compound. In instances where the fluorescence propertiesof the test molecule are not adequate for such characterizations,radiolabeled compounds are used for these assays. From the HPLC data,the rate of disappearance and/or formation of novel metabolite compoundson the lumenal surface and the appearance of test compound and/or novelmetabolite in the basolateral solution is quantitated. The data relatingthe chromatographic mobility of potential novel metabolites withreference to the parent compound are also quantitated.

[0223] To analyze the potential metabolism of test compounds by CFsputum, a “representative” mixture of expectorated CF sputum obtainedfrom 10 CF patients (under IRB approval) has been collected. The sputumhas been be solubilized in a 1:5 mixture of KBR solution with vigorousvortexing, following which the mixture was split into a “neat” sputumaliquot and an aliquot subjected to ultracentrifugation so that a“supernatant” aliquot was obtained (neat=cellular; supernatant=liquidphase). Typical studies of compound metabolism by CF sputum involve theaddition of known masses of test compound to “neat” CF sputum andaliquots of CF sputum “supernatant” incubated at 37° C., followed bysequential sampling of aliquots from each sputum type forcharacterization of compound stability/metabolism by HPLC analysis asdescribed above. As above, analysis of compound disappearance, rates offormation of novel metabolities, and HPLC mobilities of novelmetabolites are then performed.

[0224] 4. Pharmacological Effects and Mechanism of Action of the Drug inAnimals

[0225] The effect of compounds for enhancing mucociliary clearance (MCC)can be measured using an in vivo model described by Sabater et al.,Journal of Applied Physiology, 1999, pp. 2191-2196, incorporated hereinby reference.

EXAMPLES

[0226] Having generally described this invention, a furtherunderstanding can be obtained by reference to certain specific exampleswhich are provided herein for purposes of illustration only and are notintended to be limiting unless otherwise specified.

Preparation of Sodium Channel Blockers

[0227] Materials and methods. All reagents and solvents were purchasedfrom Aldrich Chemical Corp. and used without further purification. NMRspectra were obtained on either a Bruker WM 360 (¹H NMR at 360 MHz and¹³C NMR at 90 MHz) or a Bruker AC 300 (¹H NMR at 300 MHz and ¹³C NMR at75 MHz). Flash chromatography was performed on a Flash Elute™ systemfrom Elution Solution (PO Box 5147, Charlottesville, Va. 22905) chargedwith a 90 g silica gel cartridge (40M FSO-0110-040155, 32-63 μm) at 20psi (N₂). GC-analysis was performed on a Shimadzu GC-17 equipped with aHeliflex Capillary Column (Alltech); Phase: AT-1, Length: 10 meters, ID:0.53 mm, Film: 0.25 micrometers. GC Parameters: Injector at 320° C.,Detector at 320° C., FID gas flow: H₂ at 40 ml/min., Air at 400 ml/min.Carrier gas: Split Ratio 16:1, N₂ flow at 15 ml/min., N₂ velocity at 18cm/sec. The temperature program is 70° C. for 0-3 min, 70-300° C. from3-10 min, 300° C. from 10-15 min.

[0228] HPLC analysis was performed on a Gilson 322 Pump, detectorUV/Vis-156 at 360 nm, equipped with a Microsorb MV C8 column, 100 A, 25cm. Mobile phase: A=acetonitrile with 0.1% TFA, B=water with 0.1% TFA.Gradient program: 95:5 B:A for 1 min, then to 20:80 B:A over 7 min, thento 100% A over 1 min, followed by washout with 100% A for 11 min, flowrate: 1 ml/min.

Example 14-(4-Carboxymethylphenyl)butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride (9)

[0229]

Methanesulfonic Acid 4-(4-Carboxymethylphenyl)butyl Ester (6)

[0230] Compound 6 was prepared according to the published procedure¹. ¹HNMR (300 MHz, CDCl₃) δ 1.75 (m, 4H), 2.78 (m, 2H), 3.12 (s, 3H), 3.88(s, 3H), 4.22 (m, 2H), 7.28 (d, 2H), 7.98 (d, 2H).

4-(4-Carboxymethylphenyl)butylazide (7). Typical Procedure C

[0231] Compound 6 (6 g, 0.02 mol) was dissolved in 80 ml of dry DMF thensodium azide (1.8 g, 0.027 mol) was added. The suspension was stirred at80° C. (oil bath) for 3 h. The solvent was then removed at reducedpressure and the residual oil was treated with CH₂Cl₂ (100 mL). Theresulting solution was washed with water (2×100 mL), brine and driedover magnesium sulfate. The solvent was removed under reduced pressurethen the residue was redissolved in a 1:1 mixture of ethylacetate/hexanes (200 mL) and passed through a pad of silica gel. Thesolvent was removed under reduced pressure to give 4.1 g (85%) of 7 asclear oil. ¹H NMR (300 MHz, CDCl₃) δ 1.68 (m, 4H), 2.22 (t, 2H), 3.29(t, 3H), 3.92 (s, 3H), 7.28 (d, 2H) 7.98 (d, 2H).

4-(4-Carboxymethylphenyl)butylamine (8)

[0232] Azide 7 (1.7 g, 7.2 mmol) and triphenylphosphine (1.9 g, 7.2mmol) were dissolved in a 10% solution of water in THF (66 mL) andstirred overnight at 25° C. Then more triphenylphosphine (0.8 g, 3 mmol)was added and the heating was continued at 60° C. (oil bath) for 6 h.The solvent was removed under reduced pressure and the residue wastreated with 2M HCl (100 mL) and extracted with ethyl acetate (2×50 mL).The water fraction was collected and ammonium hydroxide was added untilthe pH reached approximately 13. The mixture was extracted with ethylacetate (2×100 mL) then the organic fraction was washed with brine,water and dried with sodium sulfate. Ethyl acetate was removed underreduced pressure to give 0.8 g (53%) of amine 8.

4-(4-Carboxymethylphenyl)butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride (9). Typical Procedure D

[0233] 1-(3,5-Diamino-6-chloropyrazinoyl-2-methyl-pseudothioureahydroiodide (0.2 g, 0.5 mmol) was added to a solution of 8 (0.7 g, 3.4mmol) in THF (20 mL). The reaction mixture was stirred at reflux for 6h, then the solvent was evaporated and the resultant oil was treatedwith 10% HCl (15 mL). The precipitate was isolated and crystallizedtwice from ethanol to give 9 (53 mg, 25%) as a yellow solid. ¹H NMR (300MHz, DMSO-d₆) δ 1.59 (br s, 4H), 2.71 (m, 2H), 3.83 (s, 3H), 7.40 (d,2H), 7.48 (br s, 2H), 7.80 (d, 2H), 8.92 (br s, 2H), 9.00 (br s, 1H),9.48 (br s, 2H), 10.55 (s, 1H). APCI MS m/z=420 [C₁₈H₂₂ClN₇O₃+H]⁺.

Example 24-(4-Sulfatephenyl)butylamidino-3,5-diamino-6-chloropyrazinecarboxamide(10)

[0234]

4-(4-Sulfatephenyl)butylamidino-3,5-diamino-6-chloropyrazinecarboxamide(10)

[0235]4-(4-Hydroxyphenyl)butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride 5 (0.2 g, 0.5 mmol) was dissolved in 5 mL of dry pyridineand pyridine sulfurtrioxide (450 mg, 2.5 mmol) was added. The reactionmixture was stirred overnight at room temperature and the precipitatethat formed was isolated by filtration and washed with ethyl acetate(2×25 mL) to give crude 10 (180 mg, 39%, purity 87% by HPLC). An aliquotof the crude 10 (67 mg) was purified by flash chromatography (silicagel, 6:3:0.1 methylene chloride/methanol/concentrated ammoniumhydroxide) to give 10 as a yellow solid (9.3 mg, 4% based on starting5). ¹H NMR (300 MHz, DMSO-d₆) δ 1.59 (br s, 4H), 2.58 (m, 2H), 3.28 (m,2H), 7.08 (s, 4H), 7.1-7.9 (m, 6H). ESI MS m/z=456 [C₁₆H₂₀ClN₇O₅S−H]⁻.

Example 34-[4-(2,3-Dihydroxypropyloxyl)phenylibutylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride (33)

[0236]

N-Cbz-4-(4-Hydroxyphenyl)butylamine (29)

[0237] To vigorously stirred suspension of 4 (10.5 g, 0.043 mol) in THF(approx. 150 mL) was added sodium hydrogencarbonate (11 g, 0.13 mol) andthen water until a clear solution was obtained (approx. 50 mL). Thereaction mixture was cooled to 0° C. then benzyl chloroformate (10 mL,0.07 mol) was added and the reaction was stirred overnight. The solventwas removed at reduced pressure then ethyl acetate (approx. 100 mL) wasadded to the residue. The organics were washed with HCl (2 M solution,2×30 mL), water (2×50 mL), and dried over sodium sulfate. The solventwas removed and the residue was purified by column chromatography(silica gel, 1:1 ethyl acetate/hexanes) to provide 29 (10 g, 85%) as awhite solid. ¹H NMR (300 MHz, CDCl₃) δ 1.55 (br s, 4H), 2.53 (m, 2H),3.19 (m, 2H), 5.05 (s, 2H), 5.83 (s, 1H), 6.73 (d, 2H), 7.00 (d, 2H),7.38 (m, 5H).

N-Cbz-4-(4-Allyloxyphenyl)butylamine (30)

[0238] Potassium tert-butoxide (1.7 g, 15.2 mmol) and 18-crown-6 (0.1 g,0.3 mmol) were added to a solution of 29 (4.3 g, 14.3 mmol) in dry MeCN(80 mL) and the mixture was stirred for 20 min at room temperature.After this time, allyl bromide (1.2 mL, 14.3 mmol) in MeCN (10 mL) wasadded. The reaction mixture was stirred overnight at room temperature,then the precipitate was filtered off and washed with ethyl acetate. Theorganic fractions were combined, the solvent was removed at reducedpressure and the residue was purified twice by flash chromatography(silica gel, 1:1 ethyl acetate/hexanes) to provide compound 30 (3.4 g,71%) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 1.55 (m, 4H), 2.54 (t,2H), 3.20 m, 2H), 4.50 (d, 2H), 5.08 (s, 2H), 5.28 (d, 1H), 5.40 (d,1H), 6.06 (m, 1H), 6.82 (d, 2H), 7.05 (d, 2H), 7.33 (s, 5H).

N-Cbz-4-[(2,3-Dihydroxypropyloxy)phenyl]butylamine (31)

[0239] A solution of osmium tetroxide (50 mg, 0.2 mmol) in tert-butanol(8 mL) was added to a solution of 4-methylmorpholine N-oxide monohydrate(1.2 g, 9.1 mmol) in 100 mL (1:1) acetone/water solution and the mixturewas stirred for 10 min at room temperature. After this time, 30 (3.1 g,9.0 mmol) was added in 50 mL (1:1) acetone/water solution. The reactionmixture was stirred at room temperature overnight, then NaHSO₃ (0.5 g)was added and the stirring was continued for 15 min. The acetone wasevaporated and the pH was adjusted to 5.5 by the addition of 2N HCl thenthe mixture was extracted with ethyl acetate. The organic fraction wasisolated, dried with sodium sulfate, and filtered through silica gel.Compound 31 (2.1 g, 62%) was isolated as a white solid after removingthe solvent and drying under vacuum. ¹H NMR (300 MHz, DMSO-d₆) δ 1.48(m, 2H), 1.50 (m, 2H), 2.46 (m, 2H), 3.00 (m, 2H), 3.43 (m, 2H), 3.80(m, 2H), 3.93 (t, 1H), 4.66 (d, 1H), 4.99 (s, 2H), 6.82 (d, 2H), 7.07(d, 2H), 7.26 (s, 1H), 7.33 (s, 5H).

4-[(2,3-Dihydroxypropyloxy)phenyl]butylamine (32)

[0240] Cbz-protected amine 31 (2.1 g, 5.6 mmol) was dissolved inmethanol (50 mL) and Pd/C (0.46 g, 5% wet.) was added in methanol (20mL). The reaction mixture was stirred for 3 h at 1 atmosphere ofhydrogen, then the solution was filtered through a pad of silica gel.The solvent was then evaporated to give free amine 32 (0.9 g, 66%). ¹HNMR (300 MHz, DMSO-d₆) δ 1.37 (m, 2H), 1.50 (m, 2H), 2.92 (m, 1H),3.22-4.05 (br s, 4H), 3.43 (m, 2H), 3.93 (m, 1H) 6.82 (d, 2H), 7.07 (d,2H).

4-[4-(2,3-Dihydroxypropyloxyl)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride (33).

[0241] (General Procedure Z)

[0242] 1-(3,5-Diamino-6-chloropyrazinoyl-2-methyl-pseudothioureahydroiodide (1.5 g, 3.8 mmol) was added to a solution of 32 (0.9 g, 3.7mmol) in a mixture of THF (50 mL) and diisopropylethylamine (2 mL). Thereaction mixture was stirred at reflux (66° C.) for 4 h. After thistime, the reaction mixture was cooled to room temperature and the formedprecipitate was isolated as a yellow solid. The obtained solid waswashed with 5% HCl, water, and dried under vacuum to give 33 (0.88 g) asa yellow solid. The mother liquor was evaporated and the residue waspurified by flash chromatography (silica gel, 5:1:05chloroform/methanol/concentrated ammonium hydroxide). The isolated freeamino compound was treated with 5% HCl to give an additional portion of33 (0.12 g). The total yield of 33 was 53%. ¹H NMR (300 MHz, DMSO-d₆) δ1.56 (m, 4H), 2,56 (br s, 2H), 3.31 (m, 2H), 3.42 (m, 2H), 3.82 (m, 2H),3.93 (m, 2H), 4.32 (br s, 4H), 6.84 (d, 2H), 7.10 (d, 2H), 7.45 (br s,2H), 8.81 (br s, 1H), 8.94 (br s, 1H), 9.25 (br s, 1H), 10.52 (s, 1H).APCI MS m/z=452 [C₁₉H₂₆ClN₇O₄+H]⁺.

Example 4 Synthesis of Substituted3,5-Diamino-6-chloropyrazinecarboxamide Amidines (Cont) AlternatePreparation of Amine 32 4-(4-Methoxyphenyl)butyramide (117)

[0243] 4-(4-Methoxyphenyl) butyric acid (1) (450 g, 2.32 mole) wascombined with dry THF (4 L) and 4-methylmorpholine (268 mL, 2.43 mole)in a 12 L three neck flask with mechanical stirring, an ice-methanolcooling bath and nitrogen atmosphere. Small pieces

[0244] of dry ice were used to bring the bath temperature below −20° C.Isobutyl chloroformate was added at a rate so as not to exceed aninternal temperature of −10° C. After stirring for 30 min. at −10 to−20° C., a 4.7 M solution of ammonia in methanol (990 mL, 4.64 mole) wasadded in one portion. During the addition, the reaction temperature roseto 0° C. The reaction was allowed to stir for 30 min. and then allowedto stand overnight. The product mixture was transferred to a 22 Lseparatory funnel with ethyl acetate (6 L), and 10% sodium chloridesolution (1.5 L). The layers were separated and the organic solution waswashed with 10% sodium chloride solution (4×1 L) and then brine (3×500mL). The organic layer was dried over sodium sulfate, filtered,evaporated and placed under high vacuum overnight. This afforded 432 g(97%) ofthe pure amide (117) as an offwhite solid. ¹H NMR (300 MHz,CD₃OD) δ 1.81-1.93 (m, 2H), 2.20 (t, J=7.7 Hz, 2H), 2.57 (t, J=7.7 Hz,2H), 3.74 (s, 3H), 6.82 (d, J=8.7 Hz), 7.09 (d, J=8.7 Hz, 1H). CI MSm/z=194 [CH₁₁H₁₅NO₂+H]⁺.

4-(4-Hydroxyphenyl)butylamine Hydrobromide (4)

[0245] 4-(4-Methoxyphenyl)butyramide (117) (200 g, 1.0 mole) and THF(300 mL) were combined in a 12 L three neck flask which was equippedwith a heating mantle, an internal thermometer and a reflux condenser.The suspension was slowly mechanically stirred while a 1 M BoraneTHFcomplex (1 L, 1 mole) was dripped in via a pressure equalizing additionfunnel over 20 min. Another 2.2 L (2.2 mole) of 1 M BoraneTHF complexwas dripped in over 20 min. The reaction temperature rose to 45° C.during the addition. The reaction was stirred and heated to reflux over1 h, at reflux for 2 h and then allowed to cool for 2 h. Methanol (500mL) was slowly and cautiously dripped into the reaction. Copious H₂evolution was observed. The reaction was heated at reflux for 2 h andallowed to cool overnight. The reaction was evaporated and thenco-evaporated with toluene (500 mL) to a thick oil. 48% HBr (3 L) wasslowly and cautiously added to the reaction. Bubbling and foaming wasobserved during this addition which was exothermic. After the addition,the reaction became stirrable, and was stirred at reflux for 7 h. Thereaction was allowed to cool with stirring overnight. The reaction wasstirred with ice bath cooling and then suction filtered to collect anoff white solid. The solid was co-evaporated with toluene/methanol (1:1)and then dried under vacuum at 60° C. overnight. This afforded 197 g(77%) of (4) as an off white crystalline solid. ¹H NMR (300 MHz, CD₃OD)δ 1.66 (m, 4H), 2.57 (m, 2H), 2.92 (m, 2H), 6.70 (d, J=8.5 Hz, 2H), 7.01(d, J=8.5, Hz, 2H). CI MS m/z=166 [C₁₀H₁₅NO+H]⁺.

N-Cbz-4-(4-Hydroxyphenyl)butylamine (29)

[0246] 4-(4-Hydroxyphenyl)butylamine hydrobromide (4) (197 g, 0.80mole), water (1 L), 1,4-dioxane (1 L) and sodium bicarbonate (336 g, 4mole) were combined and stirred while cooled in an ice-methanol coolingbath. Benzyl chloroformate (141 mL, 0.96 mole) was dripped in over 5min. at −220 C. with no appreciable exotherm observed. This was stirredand allowed to warm to room temperature as the cooling bath thawedovernight. An additional quantity of benzyl chloroformate (8 mL, 0.54mol) was dripped in and this was allowed to stir for 2 h. The productmixture was then evaporated to approximately 500 mL and transferred to a2 L separatory funnel with ethyl acetate while decanting away from thesolids. The aqueous layer was extracted with ethyl acetate (3×1 L). Theextracts were combined, washed with brine, dried over sodium sulfate,filtered and evaporated to afford 265 g of the crude product. A portionof the crude product (130 g) was chromatographed (silica gel, 5:1hexanes/ethyl acetate) using toluene to load the column. The remainingcrude material was crystallized from 1:1 toluene/heptane. This materialwas suction filtered to collect the solid and washed with 1:1toluene/heptane. This material was vacuum desiccated at 45° C. for 2 h.The combined yield of compound (29) was 150 g (62%) of a whitecrystalline solid. ¹H NMR (300 MHz, CDCl₃) δ 1.43-1.65 (m, 4H), 2.52 (t,J =7.4 Hz, 2H), 3.19 (q, J=6.4 Hz, 2H), 4.78 (br s, 1H), 5.09 (s, 2H),5.77 (s, 1H), 6.74 (d, J=8.5 Hz, 2H), 6.98 (d, J=8.5 Hz, 2H), 7.34 (s,5). CI MS m/z=300 [C₁₈H₂₁NO₃+H]⁺.

N-Cbz-4-[4-(2,3-Dihydroxypropyloxy)phenyl]butylamine (31)

[0247] N-Cbz-4-(4-hydroxyphenyl)butylamine (31) (30 g, 0.10 mole),glycidol (8.0 mL, 0.12 mole) ethanol (30 mL) and triethylamine (0.7 mL,0.005 mole) were stirred at reflux under argon for 2 h. The productmixture was evaporated, taken up in hot ethyl acetate and suctionfiltered through a plug of silica gel, eluting with ethyl acetate. Afterevaporating to a white solid, this solid was re-crystallized fromtoluene to afford 21.8 g (58%) of compound (31). ¹H NMR (300 MHz, CD₃OD)δ 1.42-1.65 (m, 4H), 2.54 (t, J=7.5 Hz, 2H), 3.11 (t, J=6.4 Hz, 2H),3.58-3.71 (m, 2H), 3.88-4.04 (m, 3H), 5.05 (s, 2H), 6.84 (d, J=8.7 Hz,2H), 7.06 (d, J=8.5 Hz, 2H), 7.32 (s, 5H).

4-[4-(2,3-Propanediol-1-oxy)phenyl]butylamine (32)

[0248] N-Cbz-4-[4-(2,3-dihydroxypropyloxy)phenyl]butylamine (31) (67 g,0.179 mole), ethanol (900 mL), acetic acid (50 mL) and 50% wet 10%palladium on carbon (10 g) were stirred at atmospheric pressure underH2. After stirring overnight, the reaction was purged with nitrogen andsuction filtered through a pad of celite. This was evaporated and thenco-evaporated 3 times with ethanol (500 mL). The residue waschromatographed (silica gel, 100:10:1 methylenechloride/methanol/concentrated ammonium hydroxide) to afford 38 g (89%)of pure compound (32). ¹H NMR (300 MHz, CD₃OD) δ 1.42-1.55 (m, 2H),1.55-1.68 (m, 2H), 2.56 (t, J=7.5 Hz, 2H), 2.65 (t, J=7.2 Hz, 2H),3.58-3.72 (m, 2H), 3.89-4.05 (m, 3H), 6.85 (d, J=8.7 Hz, 2H), 7.08 (d,J=8.7 Hz, 2H).

Example 54-[4-(2,3-Diacetoxypropyloxy)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamide(36)

[0249]

N-Cbz-4-[(2,3-Diacetoxypropyloxy)phenyl]butylamine (34)

[0250] Acetic anhydride (0.6 ml, 6 mmol) was added to solution of 31(0.55 g, 1.5 mmol) in dry pyridine (50 mL) under stirring. The reactionmixture was stirred 3 h at 25° C. and 3 h at 40° C. (oil bath). Afterthis time, the reaction was quenched with 2N HCl (100 mL) and extractedwith ethyl acetate. The organic fraction was washed with water and driedover sodium sulfate. The solvent was removed under reduced pressure toprovide 34 (0.6 g 86%) as a white powder. ¹H NMR (300 MHz, CDCl₃) δ 1.55(m, 4H), 1.98 (s, 3H), 2.02 (s, 3H), 2.55 (m, 2H), 4.08 (m, 2H), 4.30(m, 2H), 4.45 (m, 1H), 4.80 (br s 1H), 5.08 (s, 2H), 5.38 (m, 1H), 6.82(d, 2H), 7.08 (d, 2H), 7.35 (s, 5H).

4-[(2,3-Diacetoxypropyloxy)phenyl]butylamine (35)

[0251] Cbz-protected amine 34 (0.6 g, 1.3 mmol) was dissolved inmethanol (25 mL) containing 1% acetic acid then Pd/C (0.22 g, 5% wet.)was added. The reaction mixture was stirred for 3 h under hydrogen (1atm), then the solution was filtered through a pad of silica gel and thesolvent was evaporated to give amine 35 (0.37 g, 86%) as a clear oil.

4-[4-(2,3-Diacetoxypropyloxy)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamide(36)

[0252] 1-(3,5-Diamino-6-chloropyrazinoyl-2-methyl-pseudothioureahydroiodide (0.37g, 0.95 mmol) was added to a solution of 35 (0.27 g,0.7 mmol) in a mixture of THF (40 mL) and diisopropylethylamine (1 mL).The reaction mixture was stirred at reflux (66° C.) for 6 h. After thistime, the solvent was evaporated and the residue was dissolved in MeOH.Silica gel (25 mL) was added and the solvent was removed under reducedpressure to adsorb the compound onto the silica gel. This silica gel wasadded to the top of a silica gel column and flash chromatography (silicagel, 10:1:0.1 chloroform/methanol/concentrated ammonium hydroxide) wasperformed to obtain crude 36 (128 mg). A second chromatography gave pure36 (14 mg, 3.7%) as a yellow powder. ¹H NMR (300 MHz, DMSO-d₆) δ 1.56(m, 4H), 2.05 (s, 6H), 2.58 (m, 2H), 3.14 (m, 2H), 4.10 (m, 2H), 4.28(m, 2H), 5.28 (br s, 1H), 6.58 (br s, 2H), 6.82 (m, 2H), 7.10 m, 2H).APCI MS m/z=536[C₂₃H₃₀ClN₇O₆+H]⁺.

Example 6 4-[4-(2,2 Dimethyl-[1,3]dioxolan-4yl)methyloxyphenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamide(37)

[0253]

4-[4-(2,2 Dimethyl-[1,3]dioxolan-4yl)methyloxyphenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamide(37)

[0254] Compound 33 (150 mg, 0.3 mmol) was suspended in dry acetone (50mL) then methanol was added until a clear solution was formed (approx.15 mL). p-Toluenesulfonic acid monohydrate (25 mg) was added along withmolecular sieves (5 A) and the reaction mixture was stirred for 48 h atroom temperature. After this time, the reaction mixture was filtered,silica gel (20 mL) was added and the solvent was removed under reducedpressure. This silica gel with the reaction mixture adsorbed was addedto the top of a silica gel flash chromatography column. Compound 37 (120mg, 81%) was isolated by flash chromatography (silica gel, 10:1:0.1chloroform/methanol/concentrated ammonium hydroxide) as a yellow solid.¹H NMR (300 MHz, DMSO-d₆) δ 1.30 (s, 3H), 1.34 (s, 3H), 1.52 (br s, 4H),2.56 (br s, 2H), 3.13 (br s, 2H), 3.71 (m, 1H), 3.92 (m, 2H), 4.08 (m,1H), 4.45 (m, 1H), 6.64 (br s, 2H), 6.82 (m, 2H), 7.12 (m, 2H). APCI MSm/z=492 [C₂₂H₃₀ClN₂₂H₃₀ClN₇O₄+H]⁺.

Example 74-[4-(Methyl-2,3,4-tri-O-acetyl-glucopyranonuronate-1-O-yl)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamide(40)

[0255]

2,3,4-Tri-O-acetyl-1-O-[4-(4-benzyloxycarbonylaminobutyl)phenyl]glucopyranuronicAcid Methyl Ester (38)

[0256] 2,3,4-Tri-O-acetyl-a-D-glucuronic acid methyl estertrichloroimidate (1.6 g, 3.3 mmol) was added under argon to protectedaminophenol 29 in dry methylene chloride (40 mL) then the solution wascooled to −25° C. After stirring for 10 min, BF₃.OEt₂ (0.045 mL, 0.33mmol) was added in methylene chloride (5 mL). The reaction mixture wasstirred 1.5 h at −25° C., then allowed to warm up to −10° C. andstirring was continued 1 h at that temperature. After this time, thetemperature was increased to 25° C. and the reaction mixture was stirredfor 1 h then quenched with saturated ammonium chloride (25 mL). Themixture was extracted with methylene chloride then the organic fractionwas washed with water and dried over sodium sulfate. The solvent wasevaporated and the residue was purified by flash chromatography (silicagel, 1:2 ethyl acetate/hexanes) to provide 38 (1.5 g, 72%) as a whitesolid. ¹H NMR (300 MHz, DMSO-d₆) δ 1.41 (m, 2H), 1.51 (m, 2H), 1.99-2.02(m, 9H), 2.54 (m, 2H), 3.00 (m, 2H), 3.63 (s, 3H), 4.69 (m, 1H), 4.99(s, 2H), 5.04-5.10 (m, 2H), 5.46 (m, 1H), 5.60 (m, 1H), 6.88 (d, 2H),7.12 (d, 2H), 7.27 (m, 1H), 7.33 (s, 5H). APCI MS m/z=616[C₃₁H₃₇NO₁₂+H]⁺.

2,3,4-Tri-O-acetyl-1-O-[4-(4-aminobutyl)phenyl]glucopyranuronic AcidMethyl Ester (39)

[0257] Glucuronide 38 (1.5 g, 2.4 mmol) was dissolved in dry methanol(100 mL) and Pd/C (0.62 g, 5%) was added. The reaction mixture wasstirred under hydrogen (1 atm) for 2.5 h at room temperature. After thistime, the solution was passed through a pad of silica gel and thesolvent was evaporated under reduced pressure to give amine 39 (0.94 g,84%). ¹H NMR (300 MHz, CDCl₃) δ 1.46 (m, 2H), 1.60 (m, 2H), 2.08 (m,9H), 2.58 (m, 2H), 2.72 (m, 2H), 3.63 (s, 3H), 4.14 (m, 1H), 5.10 (m,1H), 5.34 (m, 3H), 6.90 (d, 2H), 7.12 (d, 2H).

4-[4-(Methyl2,3,4-tri-O-Acetyl-glucopyranonuronate-1-O-yl)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamide(40)

[0258] 1-(3,5-Diamino-6-chloropyrazinoyl-2-methyl-pseudothioureahydroiodide (0.30 g, 0.8 mmol) was added to a solution of 39 (0.4 g, 0.8mmol) in a mixture of THF (40 mL) and diisopropylethylamine (3 mL). Thereaction mixture was stirred at reflux (66° C.) for 2 h. After thistime, the solvent was evaporated and the residue was suspended in THF.Silica gel (15 mL) was added and the solvent was removed under reducedpressure. This silica gel was transferred onto the top of a silica gelchromatography column. The target compound 40 (0.32 g, 48%) was purifiedby flash chromatography (silica gel, 12:1:0.1chloroform/ethanol/concentrated ammonium hydroxide) and isolated as ayellow powder. ¹H NMR (300 MHz, CDCl₃) δ 1.61 (br s, 4H), 2.05 (s, 9H),2.55 (m, 2H), 3.49 (br s, 2H), 3.71 (m, 3H), 4.22 (m, 1H), 5.12 (m, 1H),5.34 (m, 3H), 6.88 (m, 2H), 7 04 (d, 2H). APCI MS m/z=694[C₂₉H₃₆ClN₇O₁₁+H]⁺.

Example 84-[4-(5-Carboxy-Glucopyranonuronate-1-O-yl)-phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamide(41)

[0259]

4-[4-(5-Carboxy-2,3,4-tri-O-acetyl-glucopyranonuronate-1-O-yl)phenylbutylamidino-3,5-diamino-6-chloropyrazinecarboxainide(41)

[0260] Compound 40 (0.3 1 g, 0.44 mmol) was added to mixture ofTHF/water (1: 1, 40 mL) and the resulting cloudy solution was cooled to−10° C. A solution of NaOH in water (4 mL of 1.24 N solution) was addedand stirring was continued at 10° C. for 1.5 h. After this time, thereaction mixture was allowed to warm up to room temperature and the THFwas removed under reduced pressure. The pH of the remaining solution wasadjusted to 6 by drop wise addition of 1N HCl. The formed precipitatewas collected by centrifligation and washed with iicold water (3×20 mL).Compound 41 (0.18 g, 75%) was isolated as a yellow powder after dryingunder vacuum for 48 h. 1H NMR (300 MHz, DMSO-d₆) δ 1.56 (br 5, 4H), 2.56(m, 2H), 3.19 (m, 2H), 3.15-3.40 (br s., 1H), 3.25 (m, 2H), 3.57 (m,1H), 4.87 (m, 1H), 5.10-5.40 (br d, 1H), 6.89 (m, 2H), 7.06 (m, 2H).APCI MS m/z=554 [C₂₉H₃₆ClN₇O₁₁+H]⁺.

Example 94-[4-(5-Carboxy-Glucopyranonuronate-1-O-yl)phenyl]butylainidino-3,5-diamino-6-chloropyrazinecarboxamideTrifluoroacetate (42)

[0261]

4-Methylphenylsulfonic Acid 4-(4-Methoxyphenyl)butyl Ester (1)

[0262] Pyridine (15 mL) was added drop wise to a cooled (0° C.) solutionof 4-(4-methoxyphenyl)butanol (10.0 g, 0.055 mol) and p-toluenesulfonylchloride (13.6 g, 0.072 mol) in dry chloroform (100 mL) under stirring.The reaction mixture was stirred overnight at room temperature. Afterthis time, the reaction was quenched with 10% HCl (300 mL) and extractedwith chloroform. The organic fraction was washed with saturated NaHCO₃,water and dried over magnesium sulfate. The solvent was removed underreduced pressure and the residue purified by flash chromatography(eluent:hexane/ethyl acetate 15:1) to provide 12.9 g (66%) of 1 as clearoil. ¹H NMR (360 MHz, CDCl₃) δ 1.61 (m, 4H), 2.44 (s, 3H), 2.52 (m, 2H),3.78 (s, 3H), 4.05 (m, 2H), 6.77 (d, 2H), 7.05 (d, 2H), 7.34 (d, 2H),7.78 (d, 2H).

4-(4-Methoxyphenyl)butylazide (2)

[0263] Sodium azide (3.07 g, 0.047 mol) was added to a solution of 1(12.9 g, 0.04 mol) in anhydrous DMF (70 mL) and the reaction mixture wasstirred 12 h at 80° C. (oil bath). Then solvent was removed at reducedpressure and the residual oil was treated with a mixture of CH₂Cl₂/ether3:1 (100 mL). The resulting solution was washed with water (2×100 mL),brine and dried over magnesium sulfate. The solvent was removed underreduced pressure and 7.6 g (95%) of 2 was obtained. The purity of 2(99%) was determined by GC and TLC (eluent:hexane/ethyl acetate 1:1),R_(f)=0.84.

4-(4-Methoxyphenyl)butylamine (3). Typical Procedure A

[0264] Lithium aluminum hydride (LAH) (55 mL of a 1.0 M solution in THF,0.055 mol) was added drop wise to a solution of 2 (7.6 g, 0.037 mol) indry THF (70 mL) at 0° C. The mixture was stirred overnight at roomtemperature in an argon atmosphere then the mixture was treated withwater (1.5 mL), then 15% NaOH (1.5 mL), then with more water (3 mL) andfiltered. The solid precipitate was washed with THF. The combinedorganic fractions were dried over magnesium sulfate and the solvent wasremoved under reduced pressure to give 6.2 g (94%) of 3. The purity of 3(99%) was determined by GC. ¹H NMR (360 MHz, DMSO-d₆) δ 1.34 (m, 2H),1.54 (m, 2H), 2.51 (m, 4H), 3.70 (s, 3H), 6.83 (d, 2H), 7.08 (d, 2H).¹³C (90 MHz, DMSO-d₆) δ 628.6, 330, 34.1, 41.5, 54.8, 113.1, 129.1,132.2, 157.3

4-(4-Hydroxyphenyl)butylamine Hydrobromide (4). Typical Procedure B

[0265] Amine 3 (2.32 g, 0.012 mol) was stirred in boiling 48% HBr (50mL) for 3 h. After the reaction was completed, argon was bubbled throughthe solution and the solvent was evaporated under reduced pressure. Thesolid residue was dried above KOH to provide 3.1 g (90%) of 4. APCI MSm/z=166[C₁₀H₁₅NO+H]⁺.

4-(4-Hydroxyphenyl)butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride (5)

[0266] 1-(3,5-Diamino-6-chloropyrazinoyl-2-methyl-pseudothioureahydroiodide (0.4 g, 1.03 mmol) was added to a suspension of4-(4-hydroxyphenyl)butylamine hydrobromide (4) (0.8 g, 32 mmol) in amixture of THF (35 mL) and triethylamine (3 mIL). The reaction mixturewas stirred in the boiling solvent for 3h, then the supernatant wasseparated and the solvent was removed under reduced pressure. The oilyresidue was washed with water (2×30 rnL), ether (3×30 mL) and then 10%HCl (40 mL) was added. The mixture was vigorously stirred for 10 minthen the yellow solid was filtered off, dried and recrystallized twicefrom ethanol to give 5 (0.18 g, 41%) as yellow solid. Purity is 98% byHPLC, retention time is 9.77 min. ¹H NMR (300 MHz, DMSO-d₆) δ 1.56 (brs, 4H), 2.48 (br s, 2H), 3.35 (m, 2H), 6.65 (d, 2), 6.95 (d, 2H), 7.50(br s, 2H), 8.75 (br s, 1H), 9.05 (br s, 1H), 9.33 (br s, 2H), 10.55 (s,1H). ¹³C NMR (75 MHz, CD₃OD) 28.7, 29.8, 35.4, 42.4, 111.2, 116.1,122.0, 130.0, 134.0, 155.0, 156.1, 156.8, 157.5, 167.0. APCI MS m/z=378[C₁₆H₂₀ClN₇O₂+H]⁺.

4-[4-(5-Carboxy-glucopyranonuronate-1-O-yl)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamideTrifluoroacetate (42)

[0267] Compound 5 (29 mg, 0.07 mmol) was dissolved in DMF (2 mL). A 100mM TRIS buffer solution, pH 7.5, containing 10 mM MgCl₂, 1.0 mMdithiothreitol, 10 mM saccharolactone, and 2 mM CMP(cytidine-mono-phosphate) was made. 176 mg of UDP-GA(uridine-di-phospho-glucuronic acid) was dissolved in the buffersolution (30 mL) and added to 600 mg of bovine liver microsomes(produced at AMRI Biocatalysis Division) in a 50 mL widemouth jar. TheDMF solution of 5 was added to initiate the reaction. The reaction wasrun at room temperature with periodic shaking by hand and was stopped bythe addition of an equal volume of MeCN after 42 h. The reactionsolution was divided into two (50 mL) centrifuge tubes and centrifugedto remove the enzyme. The precipitated enzyme was re-suspended in MeCN(40 mL) and centrifuged again. This enzyme wash was repeated 3 timesuntil the LC/MS analysis showed only trace amounts of remaining product.The supernatants were combined and the aqueous MeCN was removed undervacuum at 30° C. The resulting syrup was thinned by the addition of MeCNand fuirther dried under vacuum overnight. After drying, the syrup waspurified by RP-HPLC (Luna C18 (2) 250×21.2 mm, 51 μm) with awater/acetonitrile (both containing 0.1% TFA) gradient. The appropriatefractions were combined and dried under vacuum to yield 42 (14.8 mg,28.2%) with 98.4% purity (by HPLC analysis) as a white solid. APCI MSm/z=554 [C₂₉H₃₆ClN₇O₁₁+H]⁺, m/z=552[C₂₉H₃₆ClN₇O₁₁−H]⁻.

Example 104-[4-(1,4-Dioxapent-1-yl)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride (66)

[0268]

N-Cbz-4-[4-(1,4-Dioxapent-1-yl)phenyl]butylamine (64)

[0269] To a vigorously stirred solution of 29 (4 g, 0.013 mol) inanhydrous THF (150 mL) under nitrogen at 0° C. was added sodium hydride(60% dispersion in mineral oil, 0.64 g, 0.016 mol). The mixture wasstirred for 15 min then tetrabutylammonium iodide (0.5 g, 0.0013 mol)and 2-bromoethyl methyl ether (2.04 g, 0.015 mol) were added and themixture was stirred at room temperature overnight. The solvent wasremoved under reduced pressure and the material was purified by columnchromatography (silica gel, 10:1 methylene chloride/ethyl acetate) toprovide 64 (3.1 g, 64%). ¹H NMR (300 MHz, CDCl₃) δ 1.60 (m, 4H), 2.59(m, 2H), 3.23 (m, 2H), 3.45 (s, 3H), 3.77 (m, 2H), 4.10 (m, 2H), 5.13(s, 2H), 6.88 (d, 2H), 7.08 (d, 2H), 7.47 (s, 5H).

4-(1,4-Dioxapent-1-yl)phenylbutylamine (65)

[0270] To a solution of 64 (2.27 g, 6.4 mmol) in ethanol (60 mL) withacetic acid (1 wt. %) was added Pd/C catalyst (300 mg, 10% wet) then themixture was shaken for 18 h at 30 psi of hydrogen in a Parrhydrogenator. The pressure was released and the catalyst was filteredoff through a pad of silica gel. The solvent was removed at reducedpressure to provide 65 (1.3 g, 92%). ¹H NMR (300 MHz, CDCl₃) δ 1.60 (brs, 4H), 2.00 (s, 2H) 2.55 (br s, 2H), 2.85 (br s, 2H), 3.47 (s, 3H),3.73 (m, 2H), 4.10 (m, 2H), 6.82 (d, 2H), 7.08 (d, 2H).

4-[4-(1,4-Dioxapent-1-yl)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride (66)

[0271] 1-(3,5-Diamino-6-chloropyrazinoyl-2-methyl-pseudothioureahydroiodide (0.3 g, 0.77 mmol) was added to an anhydrous THF solution(20 mL) of 65 (0.48 g, 2.3 mmol). The reaction mixture was stirred atreflux for 11 lh then the solvent was evaporated. The residue waspurified on a Biotage system (silica gel, 10:1:0.1chloroform/methanol/concentrated ammonium hydroxide). The appropriatefractions were collected, the solvent was evaporated and the residue wastreated with 3% HCl. The yellow precipitate that formed was separatedand washed with ethyl acetate, water and dried to provide 66 (160 mg,34%) as a yellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ 1.57 (br s, 4H),2.52 (m, 4H), 3.30 (s, 3H), 3.63 (m, 2H), 4.03 (m, 2H) 6.85 (d, 2H),7.12 (d, 2H), 7.46 (br s, 2H), 8.00 (br s, 1H), 8.85 (br s, 1H), 8.99(br s, 1H), 9.32 (br s, 1H), 10.03 (s, 1H). APCI MS m/z 436[C₁₉H₂₆ClN₇O₃+H]⁺.

Example 114-(4-Hydroxymethylphenyl)butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride (68)

[0272]

4-(4-Hydroxymethylphenyl)butylamine (67)

[0273] Lithium aluminum hydride (35 mL of a 1.0 M solution in THF, 0.035mol) was added drop wise to a vigorously stirred solution of4-(4-carboxymethylphenyl)butylazide 8 (2.4 g, 0.010 mol) in dry THF (120mL) at 0° C. and stirred overnight at room temperature under a nitrogenatmosphere. To break up the complex water (1.5 mL), 15% NaOH (1.5 mL)and water (4.5 mL) were added drop wise to the cold reaction mixture.The white solid precipitate was filtered off and washed with THF. Allorganics phases were combined and evaporated. The material was purifiedby column chromatography (silica gel, 2:1:0.05chloroform/ethanol/concentrated ammonium hydroxide) to provide 67 (1.17g, 64%) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 1.15 (br s, 2H),1.54 (br s, 2H) 1.70 (br s, 2H), 2.60 (m, 4H), 3.77 (s, 1H), 4.67 (s,2H), 7.47 (s, 2H), 7.60 (s, 2H).

4-(4-Hydroxymethylphenyl)butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride (68)

[0274] 1-(3,5-Diamino-6-chloropyrazinoyl-2-methyl-pseudothioureahydroiodide (0.2 g, 0.52 mmol) was added to an anhydrous THF suspension(20 mL) of 67 (0.37 g, 2.06 mmol). The reaction mixture was stirred atreflux for 3 h then the solvent was evaporated. The residue was washedwith ethyl acetate (3×15 mL), dried and treated with 3% HCl (15 mL). Theyellow solid that formed was filtered, washed with water (2×10 mL) anddried to provide 68 (216 mg, 98%). ¹H NMR (300 MHz, DMSO-d₆) δ 1.57 (brs, 4H), 2.62 (m, 2H), 3.35 (m, 2H), 3.73 (br s, 4H), 4.45 (s, 2H), 7.12(d, 2H), 7.24 (d, 2H), 8.85 (br s, 1H), 9.98 (br s, 1H), 9.32 (br s,1H), 10.55 (s, 1H). APCI MS m/z 392 [C₁₇H₂₂ClN₇O₃+H]⁺.

Example 124-{4-1(2R)-2,3-Dihydroxypropyloxy]phenyl]}butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride (71)

[0275]

N-Cbz-4-{[(2R)-2,3-Dihydroxypropyloxy]phenyl}butylamine (69)

[0276] To cold (0° C.) N-Cbz-4-(4-allyloxyphenyl)butylamine 30 (1.94 g,5.7 mmol) under a nitrogen atmosphere was added cold (0° C.) AD-Mix-α(12 g) in tert-butanol (100 mL) and water (100 mL). The mixture wasallowed to warm to room temperature overnight. The mixture was thenquenched with saturated sodium sulfite (200 mL), extracted with ethylacetate (3×100 mL), dried (Na₂SO₄) and concentrated to give 69 (2 g,95%) as a beige solid. ¹H NMR (300 MHz, DMSO) δ 1.40 (m, 2H), 1.54 (m,2H) 2.55 (br s, 2H), 3.00 (m, 2H), 3.44 (s, 2H), 3.78 (m, 2H), 3.94 (m,1H), 4.68 (br s, 1H), 4.93 (s, 1H), 5.00 (s, 2H), 6.83 (d, 2H), 7.08 (d,2H), 7.30 (br s, 1H), 7.35 (s, 5H).

4-{[(2R)-2,3-Dihydroxypropyloxy]phenyl}butylamine (70)

[0277] To a vigorously stirred solution of 69 (2 g, 5.4 mmol) inmethanol (60 mL) under nitrogen was added Pd/C (10% wet, 0.5 g). Themixture was stirred 2 h at room temperature under an atmosphere ofhydrogen then the pressure was released and the mixture was filteredthrough a pad of silica gel. The solvent was removed and the residue waspurified by column chromatography (silica gel, 2:1:0.2chloroform/ethanol/concentrated ammonium hydroxide) to provide 70 (1.18g, 92%) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 1.32 (m, 2H), 1.54(m, 2H) 2.55 (m, 2H), 3.45 (m, 2H), 3.82 (m, 3H), 3.94 (m, 2H), 6.83 (d,2H), 7.08 (d, 2H).

4-{4-[(2R)-2,3-Dihydroxypropyloxylphenyl]}butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride (71)

[0278] 1-(3,5-Diamino-6-chloropyrazinoyl-2-methyl-pseudothioureahydroiodide (0.4 g, 1.03 mmol) was added to an anhydrous THF suspension(50 mL) of 70 (0.49 g, 2.00 mmol). The reaction mixture was stirred atreflux for 5 h then the mixture was cooled and the precipitate thatformed was collected and washed with 3% HCl (2×5 mL) then dried toprovide 71 (290 mg, 58%) as a yellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ1.57 (s, 4H), 2.55 (d, 2H), 3.35 (d, 2H), 3.90 (m, 5H), 6.82 (d, 2H),7.10 (d, 2H), 7.47 (br s, 2H), 8.75 (br s, 1H), 8.90 (br s, 1H), 10.5(s, 1H). APCI MS m/z 452 [C ₁₉H₂₆ClN₇O₄+H]⁺.

Example 134-{4-[(2S)-2,3-Dihydroxypropyloxyl]phenyl]}butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride (74)

[0279]

N-Cbz-4-{[(2S)-2,3-Dihydroxypropyloxy]phenyl}butylamine (72)

[0280] To cold (0° C.) N-Cbz-4-(4-allyloxyphenyl)butylamine 30 (1.53 g,4.5 mmol) under a nitrogen atmosphere was added cold (0° C.) AD-Mix-β(9.2 g ) in tert-butanol (100 mL) and water (100 nmL). The mixture wasallowed to warm to room temperature overnight. The mixture was quenchedwith saturated sodium sulfite (200 mL), extracted with ethyl acetate(3×100 mL), dried (Na₂SO₄) and concentrated to provide 72 (1.67 g, 99%)as a beige solid. ¹H NMR (300 MHz, CDCl₃) δ 1.54 (m, 4H) 2.55 (br s,2H), 3.18 (m, 2H), 3.70 (s, 3H), 4.02 (d, 2H), 4.10 (m, 1H), 4.73 (br s,1H), 5.08 (s, 2H), 6.83 (d, 2H), 7.08 (d, 2H), 7.38 (s, 1H), 7.35 (s,5H).

4-{[(2S)-2,3-Dihydroxypropyloxy]phenyl}butylamine (73)

[0281] Compound 73 was prepared in a similar fashion to the synthesis ofcompound 70 starting from compound 72 (1.67 g, 4.5 mmol). Amine 73 (1.06g, 99%) was isolated as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 1.32(m, 2H), 1.54 (m, 2H) 2.55 (m, 2H), 3.45 (m, 2H), 3.75 (m, 3H), 3.94 (m,2H), 6.83 (d, 2H), 7.08 (d, 2H).

4-{4-[(2S)-2,3-Dihydroxypropyloxy]phenyl]}butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride (74)

[0282] Compound 74 was prepared in a similar fashion to the synthesis ofcompound 71 starting from compound 73 (0.74 g, 3.09 mmol). Compound 74(0.38 g, 76%) was isolated as a yellow solid. ¹H NMR (300 MHz, DMSO-d₆)δ 1.57 (s, 4H), 2.55 (d, 2H), 3.35 (d, 2H), 3.85 (m, 5H), 6.82 (d, 2H),7.10 (d, 2H), 7.47 (br s, 2H), 8.75 (br s, 1H), 8.90 (br s, 1H), 10.5(s, H1H). APCI MS m/z 452 [C₁₉H₂₆ClN₇O₄+H]⁺.

Example 144-(4-Aminophenyl)ethylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride (83)

[0283]

4-(4-Aminophenyl)ethylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride(83)

[0284] A mixture of 1-H -pyrazolecarboxamidine hydrochloride (2.8 g, 19mmol), 4-aminoethylaniline (1.3 mL, 9 mmol) and diisopropylethylamine(1.3 ml) were stirred in dry DMF (5 mL) under argon for 18 h. After thistime, ether (30 ml) was added to produce a clear oil. The obtained oil(82) was washed with ether and dried under vacuum (40 mTorr) overnight.After drying 70 mg of oil was taken into dry methanol (3 mL) and 25%NaOH (0.14 mL) was added. The reaction volume was decreased to 1.0 mLand 3,5-diamino-6-chloropyrazine-2 carboxylic acid methyl ester (0.1 g,0.5 mmol) was added. The mixture was stirred at room temperatureovernight. Another portion of 82 (0.1 g) was dissolved in methanol (1mL), treated with 25% NaOH (0.15 mL) and the resulting solution wasadded to the reaction mixture. The reaction mixture was stirred 3 h atreflux, then cooled to room temperature and the solvent was removedunder reduced pressure. The residue was dissolved in a minimal volume ofDMF and separated by preparative HPLC. The obtained fractions wereanalyzed by LC/MS. The fractions containing product with M+H=349 werecollected and the solvent was removed under reduced pressure. Theresidue was dissolved in 10% HCl and evaporated to dryness to produce 83(23.5 mg, 11%) as a yellow solid. ¹H NMR (360 MHz, DMSO-d₆) δ 2.91 (m,2H), 3.59 (m, 2H), 7.31 (d, 2H), 7.42 (m, 4H), 9.02 (br s., 2H), 9.41(br s., 1H), 10.56 (s, 1H). ¹³C NMR (90 MHz, DMSO-d₆) 33.1, 42.0, 108.9,119.6, 120.7, 129.9(2C), 131.0(2C), 153.1, 154.1, 155.8, 165.2. API MSm/z=349 [C₁₄H₁₇ClN₈O+H]⁺.

[0285] Preparative HPLC was performed on a Gilson Combichem using a LunaC18(2) column, 5 μ, 250×21.2 mm; Flow rate=20 mL/min; Mobile phaseconsists of MeCN/water containing 0.1% TFA; Gradient: 10% MeCN from the0-2 min interval, concentration of MeCN increased from 10 to 40% from2-10 min, 40 to 100% MeCN from 10-19 min, 100% MeCN from 19-23 min, MeCNdecreased from 100 to 10% from 23-25 min.

Example 154-[4-(1,4,7-Trioxaoct-1-yl)phenyl]-butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride (108)

[0286]

4-[4-(1,4,7-Trioxaoct-1-yl)phenyl]-N-benzyloxycarbonylbutylamine (106)

[0287] 4-(4-Hydroxyphenyl)-N-benzyloxycarbonylbutylamine (29) (1.0 g,3.3 mmol), 1-bromo-2-(2-methoxyethoxy)ethane (0.67 g, 3.7 mmol), andpotassium carbonate (0.60 g, 4.3 mmol) were combined in acetone (20 mL),and stirred at reflux overnight. The reaction was allowed to cool, andthen filtered and evaporated. The residue was re-subjected in methylethyl ketone (10 mL), 1-bromo-2-(2-methoxyethoxy)ethane (0.91 g, 5.0mmol), potassium carbonate (0.74 g, 5.3 mmol), and sodium iodide (0.5 g,3.3 mmol) with stirring at reflux for 2.5 h. The reaction was allowed tocool, and was filtered and evaporated. The residue was re-subjected inDMF (10 mL), with 1-bromo-2-(2-methoxyethoxy)ethane (1.8 g, 9.8 mmol),potassium carbonate (1.60 g, 11.6 mmol), and sodium iodide (0.4 g, 2.7mmol), overnight with stirring at 70° C. The reaction was evaporated toremove the solvent and then was dissolved in ethyl acetate (70 mL). Theorganic extract was washed with water (3×20 mL), dried over potassiumcarbonate and filtered. Evaporation afforded 1.1 g of an oil which waspurified by column chromatography (silica gel, 2:1 hexanes/ethylacetate) to afford 800 mg (70%) of pure product 106. ¹H NMR (300 MHz,CDCl₃) δ 1.42-1.68 (m, 4H), 2.55 (t, J=7.5 Hz, 2H), 3.20 (q, J=6.2 Hz,2H), 3.39 (s, 3H), 3.55-3.60 (m, 2H), 3.69-3.74 (m, 2H), 3.82-3.87 (m,2H), 4.11 (t, 5.3 Hz, 2H), 4.71 (br s, 1H), 5.09 (s, 2H), 6.82 (d, J=8.5Hz, 2H), 7.05 (d, J=8.5 Hz, 2H), 7.34 (s, 5H). CI MS m/z=402[C₂₃H₃₁NO₅+H]⁺.

4-[4-(1,4,7-Trioxaoct-1-yl)phenyl]butylamine (107)

[0288] Compound 106 (800 mg, 2.0 mmol) in ethanol (20 mL), was subjectedto 1 atmosphere of H₂ in the presence of 10% Palladium on carbon (100mg, cat.) with stirring for 5 h. After standing overnight, the reactionwas purged with N₂ then suction filtered through celite and washed offthe celite with methylene chloride. The solvents were evaporated andchromatographed (silica gel, 200:10:1 methylenechloride/methanol/concentrated ammonium hydroxide) to afford 530 mg(>99%) of amine 107. ¹H NMR (300 MHz, CDCl₃) δ 1.31 (br s, 2H),1.41-1.52 (m, 2H), 1.55-1.67 (m, 2H), 2.56 (t, J=7.7 Hz, 2H), 2.70 (t,J=7.0 Hz, 2H), 3.39 (s, 3H), 3.58 (m, 2H), 3.72 (m, 2H), 3.84 (t, J=4.9Hz, 2H), 4.12 (t, J=5.3 Hz, 2H), 6.83 (d, J=8.7 Hz, 2H), 7.07 (d, J=8.7Hz, 2H). CI MS m/z=268 [C₁₅H₂₅NO₃+H]⁺.

4-[4-(1,4,7-Trioxaoct-1-yl)phenyl]-butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride (108)

[0289] Amine 107 (200 mg, 0.75 mmol),1-(3,5-Diamino-6-chloropyrazinoyl-2-methyl-pseudothiourea hydroiodide(296 mg, 0.76 mmol) and triethylamine (0.52 mL, 3.73 mmol) were combinedin THF (5 mL) and stirred at reflux under N₂ for 1.5 h. After stirringat room temperature for two days, the reaction was evaporated. Theresidue was chromatographed (silica gel, 400:10:1 to 200:10:1 gradientelution, methylene chloride/methanol/concentrated ammonium hydroxide) toafford the free base of the product (290 mg). This material was stirredin methanol (20 mL) at 0° C. then 1M HCl (3 mL) was added. The solutionwas evaporated with no heating and then co-evaporated with methanol. Theresidue was dissolved in methanol and then precipitated by the additionof ethyl acetate. This precipitate was centrifuged and the supernatantwas decanted. The gel like pellet was evaporated, co-evaporated withwater (2 mL) and then placed on high vacuum overnight. This afforded 129mg (42%) of compound 108 as a yellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ1.58 (m, 4H), 2.58 (br s, 2H), 3.25 (s, 3H), 3.33 (m, 2H), 3.45 (m, 2H),3.58 (m, 2H), 3.72 (m, 2H), 3.04 (m, 2H), 4.92 (br s, 4H), 6.85 (d,J=8.5 Hz, 2H), 7.12 (d, J=8.4 Hz, 2H), 8.10 to 7.26 (br m, 3H), 8.94 (brd, 2H), 9.32 (br s, 1H), M.P.=110-125° C. APCI MS m/z=480[C₂₁H₃₀ClN₇O₄+H]⁺.

Example 164-[4-(1,4,7,10,13-Pentoxatetradec-1-yl)phenyl]-butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride (112)

[0290]

O-Tosyltetraethyleneglycol Methyl Ether (109)

[0291] Tetraethyleneglycol monomethyl ether (2.0 g, 9.6 mmol) wascombined with pyridine (0.93 mL, 11.5 mmol) in methylene chloride (20mL) at 0° C. p-Toluenesulfonyl chloride (2.2 g, 11.5 mimol) dissolved inmethylene chloride (10 mL) was added dropwise and the reaction wasallowed to warm to room temperature as the ice bath thawed. Afterstirring nine days, the product mixture was transferred to a separatoryfunnel with water (70 mL). The layers were separated and the aqueouslayer was extracted with methylene chloride (3×20 mL). The extracts werecombined and evaporated. The residue (3.3 g) was chromatographed (silicagel, 4:1 to 3:1 gradient elution, methylene chloride/ethyl acetate) toafford 2.5 g (70%) of compound 109 as an oil. ¹H NMR (300 MHz, DMSO-d₆)δ 1.56 (br s, 411), 2.42 (br s, 2H), 3.34 (br s, 4H), 6.05 (s, 3H), 7.09(s, 0.5H), 7.26 (s, 0.5H), 7.42 (br s, 2H), 7.70 (br s, 2H), 8.87 (br d,2H), 9.07 (s, 2H), 9.22 (br s, 1H), 10.51 (s, 1H). CI MS m/z=363[C₁₆H₂₆O₇S+H]⁺.

4-[4-(1,4,7,10,13-Pentoxatetradec-1-yl)phenyl]-N-benzyloxycarbonylbutylamine(110)

[0292] 4-(4-Hydroxyphenyl)-N-benzyloxycarbonylbutylamine (29) (0.30 g,1.0 mmol), O-tosyltetraethyleneglycol methyl ether (109) (1.45 g, 4.0mmol), potassium carbonate (0.69 g, 5 mmol) and sodium iodide (0.6 g,4.0 mmol) were combined in DMF (5 mL) and stirred at 55° C. overnight.Cesium carbonate (0.33 g, 1.0 mmol) was added and the reaction wasstirred at 70° C. overnight. The mixture was allowed to cool and wasthen partitioned between 1:1 toluene/ethyl acetate (70 mL) and water (20mL). The layers were separated and the organic layer was washed withwater (4×10 mL), brine (2×30 mL), dried over sodium sulfate andevaporated. Chromatography (silica gel, 3:1 methylene chloride/ethylacetate) afforded 400 mg (81%) of compound 110. ¹H NMR (300 MHz, CDCl₃)δ 1.44-1.67 (m, 4H), 2.55 (t, J=7.7 Hz, 2H), 3.20 (q, J=6.0 Hz, 2H),3.37 (s, 3H), 3.54 (m, 2H), 3.61-3.75 (m, 10H), 3.84 (t, J=4.9 Hz, 2H),4.10 (t, 5.5 Hz, 2H), 4.71 (br. s, 1H), 5.09 (s, 2H), 6.82 (d, J=8.5 Hz,2H), 7.05 (d, J=8.6 Hz, 2H), 7.34 (s, 5).

4-[4-(1,4,7,10,13-Pentoxatetradec-1-yl)phenyl]butylamine (111)

[0293] This compound was prepared in a similar fashion to the synthesisof amine 107 from compound 110 (385 mg, 0.78 mmol) to give 266 mg (95%)of compound 111. ¹H NMR (300 MHz, CDCl₃) δ 1.41-1.54 (m, 2H), 1.60 (br.s, 2H), 2.56 (t, J=7.5 Hz, 2H), 2.70 (t, J=7.0 Hz, 2H), 3.37 (s, 3H),3.51-3.58 (m, 2H), 3.60-3.77 (m, 10H), 3.84 (m, 2H), 4.10 (t, 5.5 Hz,2H), 6.83 (d, J=8.3 Hz, 2H), 7.07 (d, J=8.7 Hz, 2H).

4-[4-(1,4,7,10,13-Pentoxatetradec-1-yl)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride (112)

[0294] This compound was prepared in a similar fashion to the synthesisof compound 108 from compound 111 (250 mg, 0.70 mmol) and1-(3,5-Diamino-6-chloropyrazinoyl-2-methyl-pseudothiourea hydroiodide(200 mg, 0.51 mmol) to give 130 mg (46%) of compound 112. ¹H NMR (300MHz, DMSO-d₆) δ 1.57 (br s, 4H), 2.55 (br s, 2H), 3.05 to 3.90 (m, 21H),6.85 (d, J=7.6 Hz, 2H), 7.12 (d, J=7.4 Hz, 2H), 7.44 (br s, 1H),8.10-7.40 (m, 2H), 8.93 (br d, 2H), 9.32 (s, 1H), 10.55 (s, 1H). APCI MSm/z=568 [C₂₅H₃₈ClN₇O₆S+H]⁺.

Example 174-[4-(2-Hydroxyethyloxy)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride (116)

[0295]

N-{4-[4-(2-Hydroxyethyloxy)phenyl]but-3-yn-1-yl}phthalimide (113)

[0296] 2-(4-Bromophenoxy)ethanol (3 g, 14.5 mmol), palladium (II)chloride (0.2 g, 1.1 mmol) and triphenylphosphine (0.6 g, 2.2 mmol) weredissolved in triethylamine (70 mL) then cupper(I) iodide (0.45 g, 2.1mmol) and N-(but-3-yn)phthalimide (3.2 g, 16 mmol) were added. Thereaction mixture was stirred for 72 h at room temperature and 5 h at 50°C. (oil bath), then the solvent was removed under reduced pressure.Ethyl acetate (150 mL) was added to the residue and the mixture waswashed with 2N HCl, brine and water. The organic fraction was isolated,dried with sodium sulfate and the solvent was removed under reducedpressure. The product 113 (1.7 g. 43%) was isolated by flashchromatography (silica gel, 10:1:2 methylene chloride/ethylacetate/hexanes) as a brown oil. ¹H NMR (300 MHz, CDCl₃) δ 2.80 (m, 2H),3.95 (m, 4H), 4.08 (m, 2H), 6.83 (d, 2H), 7.35 (m, 2H), 7.72 (m, 2H),7.88 (m, 2H).

N-{4-[4-(2-Hydroxyethyloxy)phenyl]butyl}phthalimide (114)

[0297] A solution of 113 (1.7 g, 5.1 mmol) in a mixture ofmethanol/ethyl acetate (80 and 10 mL correspondingly) was placed in a0.5 L Parr flask and palladium on carbon (1.1 g, 5% wet. Pd/C) wasadded. The reaction mixture was shaken at 50 psi of hydrogen pressure atroom temperature overnight. After this time, the mixture was filteredthrough a silica gel pad and the solvent was removed at reduced pressureto give crude 114 (1.2 g) as a brown oil. The crude 114 was used in thenext step without further purification.

4-[4-(2-Hydroxyethyloxy)phenyl]butylamine (115)

[0298] The crude protected amine 114 (1.2 g, 35 mmol) was dissolved in40 mL of a 2 N solution of methyl amine in dry methanol and the reactionmixture was stirred overnight at room temperature. After this time thesolvent was removed under reduced pressure and the residue was purifiedby flash chromatography (silica gel, 5:1:0.1chloroform/methanol/concentrated ammonium hydroxide) to give free amine115 (0.25 g, 35%) as a clear oil. ¹H NMR (300 MHz, DMSO-d₆) δ 1.58 (m,4H), 2.55 (m, 2H), 2.73 (m, 2H), 3.83 (m, 2H), 3.97 (m, 2H), 6.83 (d,2H), 7.07 (d, 2H), 7.82 (s, 1H).

4-[4-(2-Hydroxyethyloxy)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride (116)

[0299] 1-(3,5-Diamino-6-chloropyrazinoyl-2-methyl-pseudothioureahydroiodide (0.32 g, 0.8 mmol) was added to a solution of amine 115(0.25 g, 1.2 mmol) in a mixture of THF (15 mL), methanol (5 mL) anddiisopropylethylamine (1 mL). The reaction mixture was stirred at refluxfor 3.5 h and then cooled to room temperature. The formed precipitatewas isolated, washed with ethyl acetate (2×5 InL) and treated with 5%HCl (10 mL). The resulting solid was isolated by filtration, washed withwater and dried under vacuum to give compound 116 (0.25 g, 73%) as ayellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ 1.56 (m, 4H), 2.57 (m, 2H),3.32 (m, 2H), 3.70 (m, 2H), 3.93 (m, 2H), 4.90 (t, 1H), 6.84 (d, 2H),7.12 (d, 2H), 7.45 (s, 2H), 8.70 (br s, 1H), 8.88 (br s, 1H), 9.12 (brs, 1H) 10.45 (s, 1H). APCI MS m/z=422 [C₁₈H₂₄ClN₇O₃+H]⁺.

Example 184-[4-(2-Hydroxypropyloxy)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride

[0300]

[0301] Using general procedure Z,4-[4-(2-hydroxypropyloxy)phenyl]butylamine was converted into4-[4-(2-hydroxypropyloxy)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride, m.p. 212-214° C., APCI MS, M/Z=436 [C₁₉H₂₆ClN₇O₃+H]⁺.

Example 194-[4-(3-Hydroxypropyloxy)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride

[0302]

[0303] Using general procedure Z,4-[4-(3-hydroxypropyloxy)phenyl]butylamine was converted into4-[4-(3-hydroxypropyloxy)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride, m.p. 211-213° C., APCI MS, M/Z=436 [C₁₉H₂₆ClN₇O₃+H]⁺.

Example 204-[4-(2-{Tetrahydropyan-2-yl}oxyethyloxy)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamide

[0304]

[0305] Using general procedure Z,4-[4-(2-{Tetrahydropyan-2-yl}oxyethyloxy)phenyl]butylamine was convertedinto4-[4-(2-{Tetrahydropyan-2-yl}oxyethyloxy)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamide,m.p. 161° C., APCI Mass Spectrum, M/Z=506 [C₂₃H₃₂ClN₇O₄+H].⁺

Example 214-[3-(2-,3-Dihydroxypropyloxyl)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride

[0306]

[0307] Using general procedure Z,4-[3-(2,3-Dihydroxypropyloxy)phenyl]butylamine was converted into4-[3-(2,3-Dihydroxypropyloxy)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride, m.p. 91-93° C., APCI Mass Spectrum, M/Z=452[C₁₉H₂₆ClN₇O₄+H]⁺.

Example 224-[2-(2-,3-Dihydroxypropyloxy)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride

[0308]

[0309] Using general procedure Z,4-[2-(2,3-Dihydroxypropyloxy)phenyl]butylamine was converted into4-[2-(2,3-Dihydroxypropyloxy)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride, m.p. 200-205° C., APCI Mass Spectrum, M/Z=452[C₁₉H₂₆ClN₇O₄+H]⁺.

Example 234-[4-(2,3,4-Trihydroxybutyloxy)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride

[0310]

[0311] Using general procedure Z,4-[4(2,3,4-Trihydroxybutyloxy)phenyl]butylamine was converted into4-[4-(2,3,4-Trihydroxybutyloxy)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride. m.p. 148° C.(dec), APCI Mass Spectrum, M/Z=482 [C₂₀H₂₈ClN₇O₅+H].⁺

Example 244-[4-(4-Amino)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride

[0312]

[0313] Using general procedure Z, 4-[(4-Amino)phenyl]butylamine wasconverted into4-[4-(4-Amino)phenyl]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride. m.p. 195-200° (dec), APCI Mass Spectrum, M/Z=377[C₁₆H₂₁ClN₈O₄+H⁺].⁺

Example 254-[4-(2-Aminoethyloxy)phenyl)butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride

[0314]

[0315] Using general procedure Z,4-[4-(2{t-butoxycarbonylamino}ethyloxy)phenyl]butylamine was convertedinto4-[4-(2-{t-butoxycarbonylamino}ethyloxy)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamide,m.p. 118° C., APCI MS M/Z=521[C₂₃H₃₃ClN₈O₄+H]⁺, which was hydrolyzed andacidified with HCL to give4-[4-(2-aminoethyloxy)pheny)butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride, m.p. >178° C. (dec), M/Z=421 [C ₁₈H₂₅ClN₈O₂].

Example 264-{4-(2-Hydroxyethyl)phenyl)butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride

[0316]

[0317] Using general procedure Z,4-[4-(2-hydroxyethyl)phenyl)]butylamine was converted into4-[4-(2-hydroxyethyl)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride, m.p. 218-219° C., API M/Z =406[C₁₈H₂₄ClN₇O₂H]⁺.

Example 274-[3-(2-Hydroxyethyloxy)phenyl)butylamidino-3,5-diamino-6-chloropyrazinecarboxamideHydrochloride

[0318]

[0319] Using general procedure Z,4-[3-(2-hydroxyethyloxy)phenyl)butylamine was converted to4-[(3-(2-hydroxyethyloxy)phenyl)]butylamidino-3,5-diamino-6-chloropyrazinecarboxamidehydrochloride, m.p. 161-163° C. (dec), AMPI MS M/Z=422[C₁₈H₂₄ClN₇O₃+H].⁺

References

[0320] 1. Taylor, E. C.; Harrington, P. M.; Schin, C. Heterocycles,1989, 28, 1169

[0321] 2. Widsheis et al, Synthesis, 1994, 87-92.

Sodium Channel Blocking Activity

[0322] The compounds shown in the Tables below were tested for potencyin canine bronchial epithelia using the in vitro assay described above.Amiloride was also tested in this assay as a positive control. Theresults for the compounds of the present invention are reported asfold-enhancement values relative to amiloride.

Example 28

[0323]

Position Fold Enhancement n of R R Over Amiloride 2 4 NH₂ 12.7

Example 29

[0324]

Fold Enhancement R Over Amiloride H 124 

36 H₃CxCH₃* 91

Example 30

[0325]

n Fold Enhancement Over Amiloride 1 87 2 42 4 28.7

Example 31

[0326]

Fold Enhancement R⁵ Over Amiloride 40 65 —O—SO₃H 27.7 —O-Glucuronide11.2 Na⁺ Salt —CH₂OH 57 —CO₂CH₃ 26.5

Example 32 Effect of (Ia) on MCC

[0327] This experiment was conducted with compound (Ia), and the vehicleas a control. The results are shown in FIG. 1.

[0328] Methods

[0329] Animal Preparation: Adult ewes (ranging in weight from 25 to 35kg) were restrained in an upright position in a specialized body harnessadapted to a modified shopping cart. The animals' heads were immobilizedand local anesthesia of the nasal passage was induced with 2% lidocaine.The animals were then nasally intubated with a 7.5 mm internal diameterendotracheal tube (ETT). The cuff of the ETT was placed just below thevocal cords and its position was verified with a flexible bronchoscope.After intubation the animals were allowed to equilibrate forapproximately 20 minutes prior to initiating measurements of mucociliaryclearance.

[0330] Administration ofRadio-aerosol: Aerosols of ^(99m)Tc-Human serumalbumin (3.1 mg/ml; containing approximately 20 mCi) were generatedusing a Raindrop Nebulizer which produces a droplet with a medianaerodynamic diameter of 3.6 μm. The nebulizer was connected to adosimetry system consisting of a solenoid valve and a source ofcompressed air (20 psi). The output of the nebulizer was directed into aplastic T connector; one end of which was connected to the endotrachealtube, the other was connected to a piston respirator. The system wasactivated for one second at the onset of the respirator's inspiratorycycle. The respirator was set at a tidal volume of 500 mL, aninspiratory to expiratory ratio of 1:1, and at a rate of 20 breaths perminute to maximize the central airway deposition. The sheep breathed theradio-labeled aerosol for 5 minutes. A gamma camera was used to measurethe clearance of ^(99m)Tc-Human serum albumin from the airways. Thecamera was positioned above the animal's back with the sheep in anatural upright position supported in a cart so that the field of imagewas perpendicular to the animal's spinal cord. External radio-labeledmarkers were placed on the sheep to ensure proper alignment under thegamma camera. All images were stored in a computer integrated with thegamma camera. A region of interest was traced over the imagecorresponding to the right lung of the sheep and the counts wererecorded. The counts were corrected for decay and expressed aspercentage of radioactivity present in the initial baseline image. Theleft lung was excluded from the analysis because its outlines aresuperimposed over the stomach and counts can be swallowed and enter thestomach as radio-labeled mucus.

[0331] Treatment Protocol (Assessment of activity at t-zero): A baselinedeposition image was obtained immediately after radio-aerosoladministration. At time zero, after acquisition of the baseline image,vehicle control (distilled water), positive control (amiloride), orexperimental compounds were aerosolized from a 4 ml volume using a PariLC JetPlus nebulizer to free-breathing animals. The nebulizer was drivenby compressed air with a flow of 8 liters per minute. The time todeliver the solution was 10 to 12 minutes. Animals were extubatedimmediately following delivery of the total dose in order to preventfalse elevations in counts caused by aspiration of excess radio-tracerfrom the ETT. Serial images of the lung were obtained at 15-minuteintervals during the first 2 hours after dosing and hourly for the next6 hours after dosing for a total observation period of 8 hours. Awashout period of at least 7 days separated dosing sessions withdifferent experimental agents.

[0332] Treatment Protocol (Assessment ofActivity at t-4 hours): Thefollowing variation of the standard protocol was used to assess thedurability of response following a single exposure to vehicle control(distilled water), positive control compounds (amiloride or benzamil),or investigational agents. At time zero, vehicle control (distilledwater), positive control (amiloride), or investigational compounds wereaerosolized from a 4 ml volume using a Pari LC JetPlus nebulizer tofree-breathing animals. The nebulizer was driven by compressed air witha flow of 8 liters per minute. The time to deliver the solution was 10to 12 minutes. Animals were restrained in an upright position in aspecialized body harness for 4 hours. At the end of the 4-hour periodanimals received a single dose of aerosolized ^(99m)Tc-Human serumalbumin (3.1 mg/ml; containing approximately 20 mCi) from a RaindropNebulizer. Animals were extubated immediately following delivery of thetotal dose of radio-tracer. A baseline deposition image was obtainedimmediately after radio-aerosol administration. Serial images of thelung were obtained at 15-minute intervals during the first 2 hours afteradministration of the radio-tracer (representing hours 4 through 6 afterdrug administration) and hourly for the next 2 hours after dosing for atotal observation period of 4 hours. A washout period of at least 7 daysseparated dosing sessions with different experimental agents.

[0333] Statistics: Data were analyzed using SYSTAT for Windows, version5. Data were analyzed using a two-way repeated ANOVA (to assess overalleffects), followed by a paried t-test to identify differences betweenspecific pairs. Significance was accepted when P was less than or equalto 0.05. Slope values (calculated from data collected during the initial45 minutes after dosing in the t-zero assessment) for mean MCC curveswere calculated using linear least square regression to assessdifferences in the initial rates during the rapid clearance phase.

[0334] Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

[0335] All of the references cited above are incorporated herein byreference unless otherwise indicated.

1. A compound represented by formula (I):

wherein X is hydrogen, halogen, trifluoromethyl, lower alkyl,unsubstituted or substituted phenyl, lower alkyl-thio, phenyl-loweralkyl-thio, lower alkyl-sulfonyl, or phenyl-lower alkyl-sulfonyl; Y ishydrogen, hydroxyl, mercapto, lower alkoxy, lower alkyl-thio, halogen,lower alkyl, unsubstituted or substituted mononuclear aryl, or —N(R²)₂;R¹ is hydrogen or lower alkyl; each R² is, independently, —R⁷,—(CH₂)_(m)—OR⁸, —(CH₂)_(m)—NR⁷R¹⁰, —(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—(CH₂)_(n)—Z_(g)—R⁷, —(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂)_(n)—CO₂R⁷, or

R³ and R⁴ are each, independently, hydrogen, a group represented byformula (A), lower alkyl, hydroxy lower alkyl, phenyl, phenyl-loweralkyl, (halophenyl)-lower alkyl, lower-(alkylphenylalkyl), lower(alkoxyphenyl)-lower alkyl, naphthyl-lower alkyl, or pyridyl-loweralkyl, with the proviso that at least one of R³ and R⁴ is a grouprepresented by formula (A):

 wherein each R^(L) is, independently, —R⁷, —(CH₂)_(n)—OR⁸,—O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)—(Z)_(g)—R⁷,—O—(CH₂)_(m)—(Z)_(g)—R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronde, —O-glucose,

each o is, independently, an integer from 0 to 10; each p is an integerfrom 0 to 10; with the proviso that the sum of o and p in eachcontiguous chain is from 1 to 10; each x is, independently, O, NR¹⁰,C(═O), CHOH, C(═N—R¹⁰), CHNR⁷R¹⁰, or represents a single bond; each R⁵is, independently, —(CH₂)_(m)—OR⁸, —O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰,—O—(CH₂)_(m)—NR⁷R¹⁰, —(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)—(Z)_(g)—R⁷,—O—(CH₂)_(m)—(Z)_(g)—R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

each R⁶ is, independently, —R⁷, —OR¹¹, —N(R⁷)₂, —(CH₂)_(m)—OR⁸,—O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)—(Z)_(g)—R⁷,—O—(CH₂)_(m)—(Z)_(g)—R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)CO₂R⁷,—O—(CH₂)_(m)CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

wherein when two R⁶ are —OR¹¹ and are located adjacent to each other ona phenyl ring, the alkyl moieties of the two R⁶ may be bonded togetherto form a methylenedioxy group; each R⁷ is, independently, hydrogen orlower alkyl; each R⁸ is, independently, hydrogen, lower alkyl,—C(═O)—R¹¹, glucuronide, 2-tetrahydropyranyl, or

each R⁹ is, independently, —CO₂R⁷, —CON(R⁷)₂, —SO₂CH₃, or —C(═O)R⁷; eachR¹⁰ is, independently, —H, —SO₂CH₃, —CO₂R⁷, —C(═O)NR⁷R⁹, —C(═O)R⁷, or—CH₂—(CHOH)_(n)—CH₂OH; each Z is, independently, CHOH, C(═O), CHNR⁷R¹⁰,C═NR¹⁰, or NR¹⁰; each R¹¹ is, independently, lower alkyl; each g is,independently, an integer from 1 to 6; each m is, independently, aninteger from 1 to 7; each n is, independently, an integer from 0 to 7;each Q is, independently, C—R⁵, C—R⁶, or a nitrogen atom, wherein atmost three Q in a ring are nitrogen atoms; or a pharmaceuticallyacceptable salt thereof, and inclusive of all enantiomers,diastereomers, and racemic mixtures thereof.
 2. The compound of claim 1,wherein Y is —NH₂.
 3. The compound of claim 2, wherein R² is hydrogen.4. The compound of claim 3, wherein R¹ is hydrogen.
 5. The compound ofclaim 4, wherein X is chlorine.
 6. The compound of claim 5, wherein R³is hydrogen.
 7. The compound of claim 6, wherein each R^(L) is hydrogen.8. The compound of claim 7, wherein o is
 4. 9. The compound of claim 8,wherein p is
 0. 10. The compound of claim 9, wherein x represents asingle bond.
 11. The compound of claim 10, wherein each R⁶ is hydrogen.12. The compound of claim 11, wherein at most one Q is a nitrogen atom.13. The compound of claim 12, wherein no Q is a nitrogen atom.
 14. Thecompound of claim 13, wherein R⁵ is —(CH₂)_(m)—OR⁸.
 15. The compound ofclaim 14, which is represented by the formula:


16. The compound of claim 14, which is represented by the formula:


17. The compound of claim 13, wherein R⁵ is —O—(CH₂)_(m)—OR⁸.
 18. Thecompound of claim 17, which is represented by the formula:


19. The compound of claim 17, which is represented by the formula:


20. The compound of claim 17, which is represented by the formula:


21. The compound of claim 13, wherein R⁵ is —(CH₂)_(n)—NR⁷R¹⁰.
 22. Thecompound of claim 21, which is represented by the formula:


23. The compound of claim 13, wherein R⁵ is —O—O(CH₂)_(m)—NR⁷R¹⁰. 24.The compound of claim 23, which is represented by the formula:


25. The compound of claim 23, which is represented by the formula:


26. The compound of claim 13, wherein R⁵ is—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸.
 27. The compound of claim 13,wherein R⁵ is —O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸.
 28. The compound ofclaim 27, which is represented by the formula:


29. The compound of claim 27, which is represented by the formula:


30. The compound of claim 27, which is represented by the formula:


31. The compound of claim 27, which is represented by the formula:


32. The compound of claim 27, which is represented by the formula:


33. The compound of claim 13, wherein R⁵ is —(CH₂CH₂O)_(m)R⁸.
 34. Thecompound of claim 13, wherein R⁵ is —O—(CH₂CH₂O)_(m)—R⁸.
 35. Thecompound of claim 34, which is represented by the formula:


36. The compound of claim 34, which is represented by the formula:


37. The compound of claim 34, which is represented by the formula:


38. The compound of claim 13, wherein R⁵ is —(CH₂CH₂O))_(m)CH₂CH₂NR⁷R¹⁰.39. The compound of claim 13, wherein R⁵ is—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰.
 40. The compound of claim 13, wherein R⁵is —(CH₂)_(n)—C(═O)NR⁷R¹⁰.
 41. The compound of claim 13, wherein R⁵ is—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰.
 42. The compound of claim 13, wherein R⁵ is—(CH₂)_(n)—(Z)_(g)—R⁷.
 43. The compound of claim 13, wherein R⁵ is—O—(CH₂)_(m)—(Z)_(g)—R⁷.
 44. The compound of claim 43, which isrepresented by the formula:


45. The compound of claim 43, which is represented by the formula:


46. The compound of claim 13, wherein R⁵ is —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂O)R⁸.
 47. The compound of claim 13, wherein R⁵ is—O—(CH₂)_(m)NR¹⁰CH₂(CHOR⁸) (CHOR⁸)_(n)—CH₂O)R⁸.
 48. The compound ofclaim 13, wherein R⁵ is —O—(CH₂)_(m)—CO₂R⁷.
 49. The compound of claim13, wherein R⁵ is —OSO₃H.
 50. The compound of claim 13, wherein R⁵ is—O-glucuronide.
 51. The compound of claim 13, wherein R⁵ is —O-glucose.52. The compound of claim 13, wherein R⁵ is


53. The compound of claim 52, which is represented by the formula:


54. The compound of claim 13, wherein R⁵ is


55. The compound of claim 13, wherein R⁵ is


56. The compound of claim 55, which is represented by the formula:


57. The compound of claim 1, wherein X is halogen; Y is —N(R⁷)₂; R¹ ishydrogen or C₁-C₃ alkyl R² is —R⁷, —(CH₂)_(m)—OR⁸, or —(CH₂)_(m)—CO₂R⁷;R³ is a group represented by formula (A); and R⁴ is hydrogen, a grouprepresented by formula (A), or lower alkyl.
 58. The compound of claim57, wherein X is chloro or bromo; Y is —N(R⁷)₂; R² is hydrogen or C₁-C₃alkyl; at most three R⁶ are other than hydrogen as defined above; atmost three R^(L) are other than hydrogen as defined above; and at most 2Q are nitrogen atoms.
 59. The compound of claim 58, wherein Y is —NH₂.60. The compound of claim 59, wherein R⁴ is hydrogen; at most one R^(L)is other than hydrogen as defined above; at most two R⁶ are other thanhydrogen as defined above; and at most 1 Q is a nitrogen atom.
 61. Thecompound of claim 1, wherein R⁵ is —(CH₂)_(m)—OR⁸.
 62. The compound ofclaim 1, wherein R⁵ is —O—(CH₂)_(m)—OR⁸.
 63. The compound of claim 1,wherein R⁵ is —(CH₂)_(n)—NR⁷R¹⁰.
 64. The compound of claim 1, wherein R⁵is —O—(CH₂)_(m)—NR⁷R¹⁰.
 65. The compound of claim 1, wherein R⁵ is—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸.
 66. The compound of claim 1,wherein R⁵ is —O—(CH₂)_(m)(CHOR⁸) (CHOR⁸)_(n)—CH₂OR⁸.
 67. The compoundof claim 1, wherein R⁵ is —(CH₂CH₂O)_(m)—R⁸.
 68. The compound of claim1, wherein R⁵ is —O—(CH₂CH₂O)_(m)—R⁸.
 69. The compound of claim 1,wherein R⁵ is (CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰.
 70. The compound of claim 1,wherein R⁵ is —O(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰.
 71. The compound of claim 1,wherein R⁵ is —(CH₂)_(n)—C(═O)NR⁷R¹⁰.
 72. The compound of claim 1,wherein R⁵ is —O—(CH₂)_(m)—C(═O)NR⁷R¹⁰.
 73. The compound of claim 1,wherein R⁵ is —(CH₂)_(n)—(Z)_(g)—R.⁷
 74. The compound of claim 1,wherein R⁵ is —O—(CH₂)_(m)—(Z)_(g)—R⁷.
 75. The compound of claim 1,wherein R⁵ is —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸) (CHOR⁸)_(n)—CH₂O)R⁸.
 76. Thecompound of claim 1, wherein R⁵ is —O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸.
 77. The compound of claim 1, wherein R⁵ is—O—(CH₂)_(m)—CO₂R⁷.
 78. The compound of claim 1, wherein R⁵ is —OSO₃H.79. The compound of claim 1, wherein R⁵ is —O-glucuronide.
 80. Thecompound of claim 1, wherein R⁵ is —O-glucose.
 81. The compound of claim1, wherein R⁵ is


82. The compound of claim 1, wherein R⁵ is


83. The compound of claim 1, wherein R⁵ is


84. The compound of claim 1, wherein x is a single bond.
 85. Thecompound of claim 1, which is in the form of a pharmaceuticallyacceptable salt.
 86. A pharmaceutical composition, comprising thecompound of claim 1 and a pharmaceutically acceptable carrier.
 87. Amethod of promoting hydration of mucosal surfaces, comprising:administering an effective amount of the compound of claim 1 to amucosal surface of a subject.
 88. A method of restoring mucosal defense,comprising: topically administering an effective amount of the compoundof claim 1 to a mucosal surface of a subject in need thereof.
 89. Amethod of blocking sodium channels, comprising: contacting sodiumchannels with an effective amount of the compound of claim
 1. 90. Amethod of treating chronic bronchitis, comprising: administering aneffective amount of the compound of claim 1 to a subject in needthereof.
 91. A method of treating cystic fibrosis, comprising:administering an effective amount of the compound of claim 1 to asubject in need thereof.
 92. A method of treating sinusitis, comprising:administering an effective amount of the compound of claim 1 to asubject in need thereof.
 93. A method of treating vaginal dryness,comprising: administering an effective amount of the compound of claim 1to the vaginal tract of a subject in need thereof.
 94. A method oftreating dry eye, comprising: administering an effective amount of thecompound of claim 1 to the eye of a subject in need thereof.
 95. Amethod of promoting ocular hydration, comprising: administering aneffective amount of the compound of claim 1 to the eye of a subject. 96.A method of promoting corneal hydration, comprising: administering aneffective amount of the compound of claim 1 to the eye of a subject. 97.A method of promoting mucus clearance in mucosal surfaces, comprising:administering an effective amount of the compound of claim 1 to amucosal surface of a subject.
 98. A method of treating Sjogren'sdisease, comprising: administering an effective amount of the compoundof claim 1 to a subject in need thereof.
 99. A method of treating distalintestinal obstruction syndrome, comprising: administering an effectiveamount of the compound of claim 1 to a subject in need thereof.
 100. Amethod of treating dry skin, comprising: administering an effectiveamount of the compound of claim 1 to the skin of a subject in needthereof.
 101. A method of treating esophagitis, comprising:administering an effective amount of the compound of claim 1 to asubject in need thereof.
 102. A method of treating dry mouth(xerostomia), comprising: administering an effective amount of thecompound of claim 1 to the mouth of a subject in need thereof.
 103. Amethod of treating nasal dehydration, comprising: administering aneffective amount of the compound of claim 1 to the nasal passages of asubject in need thereof.
 104. The method of claim 103, wherein the nasaldehydration is brought on by administering dry oxygen to the subject.105. A method of preventing ventilator-induced pneumonia, comprising:administering an effective amount of the compound of claim 1 to asubject on a ventilator.
 106. A method of treating asthma, comprising:administering an effective amount of the compound of claim 1 to asubject in need thereof.
 107. A method of treating primary ciliarydyskinesia, comprising: administering an effective amount of thecompound of claim 1 to a subject in need thereof.
 108. A method oftreating otitis media, comprising: administering an effective amount ofthe compound of claim 1 to a subject in need thereof.
 109. A method ofinducing sputum for diagnostic purposes, comprising: administering aneffective amount of the compound of claim 1 to a subject in needthereof.
 110. A method of treating chronic obstructive pulmonarydisease, comprising: administering an effective amount of the compoundof claim 1 to a subject in need thereof.
 111. A method of treatingemphysema, comprising: administering an effective amount of the compoundof claim 1 to a subject in need thereof.
 112. A method of treatingpneumonia, comprising: administering an effective amount of the compoundof claim 1 to a subject in need thereof.
 113. A method of treatingconstipation, comprising: administering an effective amount of thecompound of claim 1 to a subject in need thereof.
 114. The method ofclaim 113, wherein the compound is administered orally or via asuppository or enema.
 115. A method of treating chronic diverticulitis,comprising: administering an effective amount of the compound of claim 1to a subject in need thereof.
 116. A method of treating rhinosinusitis,comprising: administering an effective amount of the compound of claim 1to a subject in need thereof.
 117. A composition, comprising: thecompound of claim 1; and a P2Y2 inhibitor.
 118. A composition,comprising: the compound of claim 1; and a bronchodilator.
 119. Thecompound of claim 1, wherein R⁵ is selected from the group consisting of—O—(CH₂)₃—OH, —NH₂, —O—CH₂—(CHOH)₂—CH₂OH—O—CH₂—CHOH—CH₂OH,—O—CH₂CH₂—O-tetrahydropyran-2-yl, —O—CH₂CHOH—CH₂—O-glucuronide,—O—CH₂CH₂OH, —O—(CH₂CH₂O))₄—CH₃, —O—CH₂CH₂OCH₃,—O—CH₂—(CHOC(═O)CH₃)—CH₂—OC(═O)CH₃, —O—(CH₂CH₂O))₂—CH₃,—OCH₂—CHOH—CHOH—CH₂OH, —CH₂OH, —CO₂CH₃,


120. The compound of claim 1, wherein R⁵ is selected from the groupconsisting of para —O—(CH₂)₃—OH, para —NH₂, para —O—CH₂—(CHOH)₂—CH₂OH,ortho —O—CH₂—CHOH—CH₂OH, meta —O—CH₂—CHOH—CH₂OH, para—O—CH₂CH₂—O-tetrahydropyran-2-yl, para —O—CH₂CHOH—CH₂—O-glucuronide,para —O—CH₂CH₂OH, para —O—(CH₂CH₂O)₄—CH₃, para —O—CH₂CH₂OCH₃, para—O—CH₂—(CHOC(═O)CH₃) —CH₂—OC(═O)CH₃, para —O—(CH₂CH₂O)₂—CH₃,—OCH₂—CHOH—CHOH—CH₂OH, para —CH₂OH, para —CO₂CH₃, para —SO₃H, para—O-glucuronide, para


121. The compound of claim 119, wherein X is chloro or bromo; Y is—N(R⁷)₂; R¹ is hydrogen or C₁-C₃ alkyl; R² is hydrogen or C₁-C₃ alkyl;R³ is a group represented by formula (A); and R⁴ is hydrogen, a grouprepresented by formula (A), or lower alkyl; at most three R⁶ are otherthan hydrogen as defined above; at most three R^(L) are other thanhydrogen as defined above; and at most 2 Q are nitrogen atoms.
 122. Thecompound of claim 121, wherein R⁴ is hydrogen; at most one R^(L) isother than hydrogen as defined above; at most two R⁶ are other thanhydrogen as defined above; and at most 1 Q is a nitrogen atom.
 123. Thecompound of claim 120, wherein X is chloro or bromo; Y is —N(R⁷)₂; R¹ ishydrogen or C₁-C₃ alkyl; R² is hydrogen or C₁-C₃ alkyl; R³ is a grouprepresented by formula (A); and R⁴ is hydrogen, a group represented byformula (A), or lower alkyl; at most three R⁶ are other than hydrogen asdefined above; at most three R^(L) are other than hydrogen as definedabove; and at most 2 Q are nitrogen atoms.
 124. The compound of claim123, wherein R⁴ is hydrogen; at most one R^(L) is other than hydrogen asdefined above; at most two R⁶ are other than hydrogen as defined above;and at most 1 Q is a nitrogen atom.